U.S. patent application number 12/911091 was filed with the patent office on 2011-10-27 for methods and kits used in identifying glioblastoma.
This patent application is currently assigned to THE TRANSLATIONAL GENOMICS RESEARCH INSTITUTE. Invention is credited to NHAN TRAN.
Application Number | 20110262426 12/911091 |
Document ID | / |
Family ID | 43900719 |
Filed Date | 2011-10-27 |
United States Patent
Application |
20110262426 |
Kind Code |
A1 |
TRAN; NHAN |
October 27, 2011 |
METHODS AND KITS USED IN IDENTIFYING GLIOBLASTOMA
Abstract
The invention encompasses methods and kits used in the
identification of invasive glioblastoma based upon the expression
of TROY. The methods and kits also allow prediction of disease
outcome as well as therapeutic outcome.
Inventors: |
TRAN; NHAN; (PHOENIX,
AZ) |
Assignee: |
THE TRANSLATIONAL GENOMICS RESEARCH
INSTITUTE
PHOENIX
AZ
|
Family ID: |
43900719 |
Appl. No.: |
12/911091 |
Filed: |
October 25, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61254615 |
Oct 23, 2009 |
|
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Current U.S.
Class: |
424/130.1 ;
435/6.12; 435/6.14; 435/7.1; 435/7.9; 506/9; 514/393 |
Current CPC
Class: |
G01N 33/574 20130101;
A61K 2039/505 20130101; G01N 33/57411 20130101; A61K 39/39558
20130101; C12Q 1/6886 20130101; A61K 31/00 20130101; G01N 2800/52
20130101; A61K 31/495 20130101; A61K 38/00 20130101; G01N 2800/54
20130101; A61K 31/713 20130101; A61P 35/00 20180101 |
Class at
Publication: |
424/130.1 ;
435/7.1; 435/6.14; 435/7.9; 435/6.12; 506/9; 514/393 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12Q 1/68 20060101 C12Q001/68; A61P 35/00 20060101
A61P035/00; C40B 30/04 20060101 C40B030/04; A61K 31/4188 20060101
A61K031/4188; G01N 33/566 20060101 G01N033/566; G01N 21/64 20060101
G01N021/64 |
Claims
1. A method of identifying a tumor as invasive glioblastoma;
comprising: adding a first reagent capable of binding to a marker
selected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 2
to a mixture comprising a sample of the tumor; subjecting the
mixture to conditions that allow detection of the binding of the
reagent to the marker; and classifying the tumor into a cohort
selected from the group consisting of invasive glioblastoma and
proliferative glioblastoma on the basis of a result of the binding
of the reagent to the sample.
2. The method of claim 1 wherein the marker includes SEQ ID NO.
2.
3. The method of claim 2 wherein the first reagent comprises a
first antibody.
4. The method of claim 3 wherein the first antibody comprises a
first label.
5. The method of claim 4 wherein the first label comprises a
fluorescent compound.
6. The method of claim 4 wherein the first label comprises an
enzyme.
7. The method of claim 4 wherein the first label comprises a
radioisotope.
8. The method of claim 4 wherein the first label comprises a
ligand.
9. The method of claim 3 further comprising adding a second
antibody to the mixture, wherein the second antibody is capable of
binding to the first antibody.
10. The method of claim 8 wherein the second antibody comprises a
second label.
11. The method of claim 1 wherein the marker includes SEQ ID NO.
1.
12. The method of claim 11 wherein the first reagent comprises a
first nucleic acid.
13. The method of claim 12 wherein the first nucleic acid comprises
a first oligonucleotide capable of binding to part of the
marker.
14. The method of claim 13 further comprising purifying RNA from
the sample, performing reverse transcription on the RNA, and adding
a second oligonucleotide capable of binding to part of the marker
to the mixture, wherein the conditions comprise nucleic acid
amplification, wherein the first oligonucleotide and the second
oligonucleotide are capable of binding to different sequences on
the marker and wherein the first oligonucleotide and the second
oligonucleotide are capable of binding to separate nucleic acid
strands.
15. The method of claim 14 wherein the first oligonucleotide
includes SEQ ID NO. 3.
16. The method of claim 14 wherein the second oligonucleotide
includes SEQ ID NO. 4.
17. The method of claim 14 further comprising adding a third
oligonucleotide to the mixture wherein the third oligonucleotide is
capable of binding to a part of the marker between the sequences to
which the first oligonucleotide and the second oligonucleotide are
capable of binding.
18. The method of claim 17 wherein the third oligonucleotide
comprises a fluorescent compound.
19. The method of claim 18 wherein the fluorescent compound is
selected from the group consisting of FAM, dR110, 5-FAM, 6FAM,
dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED, dROX, PET, and
LIZ.
20. The method of claim 18 further comprising performing DNA
sequencing on a product of the nucleic acid amplification.
21. The method of claim 1 wherein the first reagent is affixed to a
solid substrate.
22. The method of claim 21 wherein the conditions comprise
microarray analysis.
23. The method of claim 1 wherein the sample comprises a brain
biopsy.
24. A method of predicting disease outcome of a patient with
glioblastoma; comprising: adding a first reagent capable of binding
to a marker selected from the group consisting of SEQ ID NO. 1 and
SEQ ID NO. 2 to a mixture comprising a sample from the patient;
subjecting the mixture to conditions that allow detection of the
binding of the reagent to the marker; and classifying the patient
into a cohort on the basis of a result of the binding of the
reagent to the sample; wherein the cohort is selected from the
group consisting of short term survivors and long term
survivors.
25. The method of claim 24 wherein short term survivors are
predicted to survive less than 680 days.
26. The method of claim 24 wherein short term survivors are
predicted to survive less than 400 days.
27. The method of claim 24 wherein long term survivors are
predicted to survive more than 680 days.
28. The method of claim 24 wherein long term survivors are
predicted to survive more than 950 days.
29. A method of treating a patient with glioblastoma; comprising
adding a first reagent capable of binding to a marker selected from
the group consisting of SEQ ID NO. 1 and SEQ ID NO. 2 to a mixture
comprising a sample from the patient; subjecting the mixture to
conditions that allow detection of the binding of the reagent to
the marker; and treating the patient on the basis of a result of
the binding of the reagent to the sample.
30. The method of claim 29 wherein the result comprises expression
of the marker below a threshold and wherein treating the patient
comprises administering a therapeutic composition comprising a
compound selected from the group consisting of temozolimide and
bevacizumab.
31. The method of claim 29 wherein the result comprises expression
of the marker above a threshold and wherein treating the patient
comprises administering a therapeutic composition comprising a
compound selected from the group consisting of TROY inhibitor, Pyk2
inhibitor, Rac1 inhibitor, Dock180 inhibitor, and Dock7
inhibitor.
32. A kit used to identify a tumor as invasive glioblastoma
comprising: a first reagent capable of specific binding to a marker
selected from the group consisting of SEQ ID NO. 1 and SEQ ID NO.
2; and an indication of a result, wherein the result signifies that
the tumor is an invasive glioblastoma.
33. The kit of claim 32 wherein the first reagent comprises a first
antibody.
34. The kit of claim 33 wherein the first antibody comprises a
first label.
35. The kit of claim 33 further comprising a second antibody
capable of binding to the first antibody.
36. The kit of claim 35 wherein the second antibody comprises a
second label.
37. The kit of claim 33 wherein the reagent comprises a first
nucleic acid.
38. The kit of claim 37 wherein the first nucleic acid comprises a
first oligonucleotide capable of binding to part of the marker.
39. The kit of claim 38 wherein the first oligonucleotide is
selected from the group consisting of: SEQ ID NO. 3 and SEQ ID NO.
4.
40. The kit of claim 32 further comprising an enzyme.
41. The kit of claim 40 wherein the enzyme comprises a DNA
polymerase.
42. The kit of claim 40 wherein the enzyme comprises a reverse
transcriptase.
43. The kit of claim 32 wherein the first reagent is affixed to a
solid substrate.
44. The kit of claim 32 wherein the indication comprises a positive
control.
45. The kit of claim 32 wherein the indication is physically
included in the kit.
46. The kit of claim 32 wherein the indication comprises a
writing.
47. The kit of claim 46 wherein the writing is made available via a
website.
48. The kit of claim 46 wherein the writing comprises a photograph.
Description
BACKGROUND OF THE INVENTION
[0001] Glioblastoma multiforme (GBM) is the most malignant form of
all primary adult brain tumors (See Reference 1) Although
significant technical advances in surgical and radiation treatment
for brain tumors have emerged, their impact on clinical outcome for
patients has been only modest (See References 2-4). Of the features
that characterize GBM, arguably none is more clinically significant
than the propensity of glioma cells to infiltrate into normal brain
tissue. These invasive cells render complete resection impossible
and confer resistance to chemo- and radiation-therapy. Thus,
improved treatment of malignant glioma awaits a way of targeting
the dispersing tumor cells in the CNS.
BRIEF SUMMARY OF THE INVENTION
[0002] It is an object of the invention to identify a tumor as an
invasive glioblastoma.
[0003] It is an object of the invention to predict the survival
time of a patient with glioblastoma.
[0004] It is an object of the invention to treat patients with
glioblastoma.
[0005] It is an object of the invention to provide kits that
facilitate the identification of invasive glioblastoma.
[0006] It is an object of the invention to provide a personalized
medicine based test used in staging glioblastoma patients for
treatment.
[0007] The above and other objects may be achieved through the use
of methods involving, obtaining a sample of a tumor from a subject,
adding a first reagent capable of binding to a marker selected from
the group consisting of SEQ ID NO. 1 and SEQ ID NO. 2 to a mixture
comprising the sample; subjecting the mixture to conditions that
allow detection of the binding of the reagent to the marker; and
classifying the tumor into a cohort selected from the group
consisting of invasive glioblastoma and proliferative glioblastoma
on the basis of a result of the binding of the reagent to the
sample. In some aspects of the invention, the marker comprises SEQ
ID NO. 2. In those aspects, the first reagent may comprise a first
antibody. The first antibody may comprise a first label. The first
label may be any label, such as a fluorescent compound, an enzyme,
a radioisotope, or a ligand. The method may further comprise adding
a second antibody capable of binding to the first antibody to the
mixture. The second antibody may comprise a second label. The
second label may be any label such as a fluorescent compound, an
enzyme, a radioisotope, or a ligand.
[0008] In other aspects of the invention, the marker may comprise
SEQ ID NO. 1. In those other aspects, the first reagent may
comprise a first nucleic acid and the first nucleic acid may
further comprise a first oligonucleotide capable of binding to part
of SEQ ID NO. 1. The method may further comprise purifying RNA from
the sample, performing reverse transcription on the RNA, adding a
second oligonucleotide capable of binding to part of SEQ ID NO. 1
to the mixture, wherein the conditions comprise subjecting the
mixture to nucleic acid amplification, wherein the first
oligonucleotide and the second oligonucleotide are capable of
binding to different sequences on SEQ ID NO. 1, and wherein the
first oligonucleotide and the second oligonucleotide are capable of
binding to separate nucleic acid strands. The first oligonucleotide
may be any oligonucleotide such as an oligonucleotide that includes
SEQ ID NO. 3. The second oligonucleotide may be any oligonucleotide
including an oligonucleotide that includes SEQ ID NO. 4. The method
may further comprise adding a third oligonucleotide to the mixture,
wherein the third oligonucleotide is capable of binding to part of
SEQ ID NO. 1 between the sequences to which the first
oligonucleotide and the second oligonucleotide are capable of
binding. The third oligonucleotide may comprise a fluorescent
compound. The fluorescent compound may be any fluorescent compound
including a compound selected from the group consisting of FAM,
dR110, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED,
dROX, PET, BHQ+, Gold540, and LIZ. The method may comprise
performing DNA sequencing on a product of the nucleic acid
amplification. In some aspects of the invention, the first reagent
may be affixed to a solid substrate. In those aspects, the
conditions may comprise microarray analysis. The sample may be any
sample such as a sample that comprises a cell. One example of such
a sample is a brain biopsy sample.
[0009] The above and other objects may be achieved through the use
of methods involving obtaining a sample of a tumor from a patient,
adding a first reagent capable of binding to a marker selected from
the group consisting of SEQ ID NO. 1 and SEQ ID NO. 2 to a mixture
comprising the sample, subjecting the mixture to conditions that
allow detection of the binding of the reagent to the marker, and
classifying the patient into a cohort selected from the group
consisting of short term survivors and long term survivors. The
short term survivors may be predicted to survive less than 680 days
from biopsy including less than 400 days from biopsy. The long term
survivors may be predicted to survive more than 680 days from
biopsy, including more than 950 days from biopsy.
[0010] The above and other objects may be achieved through the use
of methods involving: obtaining a sample of a tumor from a patient,
adding a first reagent capable of binding to a marker selected from
a group consisting of SEQ ID NO. 1 and SEQ ID NO. 2 to a mixture
comprising the sample, subjecting the mixture to conditions that
allow detection of the binding of the reagent to the marker, and
treating the patient on the basis of a result of the binding of the
reagent to the sample. The result may be the expression of the
marker below a threshold level and treating the patient may
comprise administering a therapeutic composition comprising a
compound selected from the group consisting of temozolimide and
bevacizumab. The result may be expression of the marker above a
threshold level and treating the patient may comprise administering
a therapeutic composition comprising a compound selected from the
group consisting of TROY inhibitor, Pyk2 inhibitor, Rac1 inhibitor,
Dock180 inhibitor, and Dock7 inhibitor.
[0011] The above and other objects may be achieved through the use
of kits involving a first reagent capable of specific binding to a
marker selected from the group consisting of SEQ ID NO. 1 and SEQ
ID NO. 2 and an indication of a result, wherein the result
signifies that the tumor is an invasive glioblastoma. The first
reagent may comprise a first antibody. The first antibody may
comprise a first label. The first label may be any label such as a
fluorescent compound, an enzyme, a radioisotope, or a ligand. The
kit may further comprise a second antibody capable of binding to
the first antibody. The second antibody may comprise a second
label. The second label may be any label such as a fluorescent
compound, an enzyme, a radioisotope, or a ligand. The first reagent
may comprise a first nucleic acid. The first nucleic acid may
comprise a first oligonucleotide capable of binding to part of SEQ
ID NO. 1. The first oligonucleotide may be any oligonucleotide
meeting this description, including SEQ ID NO. 3 and SEQ ID NO. 4.
The kit may further comprise a second oligonucleotide capable of
binding to part of SEQ ID NO. 1 wherein the first oligonucleotide
and the second oligonucleotide are capable of binding to different
sequences on SEQ ID NO. 1 and wherein the first oligonucleotide and
the second oligonucleotide are capable of binding to separate
nucleic acid strands. The kit may further comprise a third
nucleotide wherein the third nucleotide binds to part of SEQ ID NO.
1 between the sequences to which the first oligonucleotide and the
second oligonucleotide are capable of binding. The third nucleic
acid may comprise a fluorescent compound. The florescent compound
may be any fluorescent compound including dR110, 5-FAM, 6FAM, dR6G,
JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED, dROX, PET, BHQ+, Gold540,
and LIZ. The kit may further comprise an enzyme. The enzyme may be
any enzyme such as a DNA polymerase or a reverse transcriptase. The
first reagent may be affixed to a solid substrate. The indication
may be any indication, such as a positive control or a writing. A
writing may be made available via a website, or it may include a
photograph. The indication may be physically included in the kit.
The indication may comprise software configured to detect a level
of expression as input and identification of invasive glioblastoma
as output. The software may be incorporated into a machine
configured to detect binding of the reagent to the marker.
BRIEF DESCRIPTION OF THE FIGURES
[0012] A more complete understanding of the present invention may
be derived by referring to the detailed description when considered
in connection with the following illustrative figures.
[0013] FIG. 1 depicts expression of TROY in normal brain and
various glioblastoma types in the NCBI Gene Expression Omnibus
GDS1962 dataset.
[0014] FIG. 2 depicts expression of TROY in a separate set of
normal brain and various glioblastoma types by QRT-PCR.
[0015] FIG. 3 depicts TROY expression data from the NCBI Gene
Expression Omnibus GDS1962 dataset with tissues grouped as
long-term and short-term survivors.
[0016] FIG. 4 depicts a Western blot showing expression of TROY in
four glioblastoma cell lines.
[0017] FIG. 5 depicts a Western blot showing suppression of TROY
expression in two of the cell types using siRNA targeting TROY.
[0018] FIG. 6 depicts a graph showing significantly slowed
migration of four glioblastoma cell lines when those lines are
transfected with siRNA targeting TROY.
[0019] FIG. 7 depicts expression of a construct comprising
HA-tagged TROY transfected into two glioblastoma cell lines.
[0020] FIG. 8 depicts increased migration rate of cell lines
transfected with the HA-tagged TROY construct.
[0021] FIG. 9 depicts increased depth of invasion into rat brain
slices by a GFP-tagged TROY transfected cell line.
[0022] FIG. 10 depicts immunofluorescent staining for HA in
HA-tagged TROY transfected cell lines.
[0023] FIG. 11 depicts (top) a Western Blot showing cell lysates
from a glioblastoma cell line transfected with Pyk2 or HA-tagged
TROY as indicated and (bottom) a Western blot of immunoprecipitates
with anti-HA antibodies that show an association of Pyk2 with
TROY.
[0024] FIG. 12 depicts increased migration rate in a glioblastoma
cell line transfected with HA-tagged TROY that is slowed when Pyk2
expression is suppressed.
[0025] FIG. 13 depicts reduced migration rate in a glioblastoma
cell line transfected with a dominant-negative Pyk2 construct,
whether or not the TROY expression is endogenous or from HA-tagged
TROY.
[0026] FIG. 14 depicts a Western blot showing increased
phosphorylation of RhoA when TROY expression is suppressed and
reduced phosphorylation of Rac-1 when TROY expression is
suppressed.
[0027] FIG. 15 depicts a Western blot showing increased
phosphorylation of Rac-1 when a cell line is transfected with
HA-tagged TROY. This effect is reduced when Pyk2 expression is
suppressed.
[0028] FIG. 16 depicts reduced migration of a glioblastoma cell
line when Rac-1 expression is suppressed--whether or not the cell
line expresses endogenous or HA-tagged TROY.
[0029] FIG. 17 depicts a Western blot validation of suppression of
Rac1 expression by Rac1 siRNA.
[0030] FIG. 18 depicts increased Akt, IkBa, and Erk1/2
phosphorylation when a glioblastoma cell line is transfected with
HA-tagged TROY.
[0031] FIG. 19 depicts increased sensitivity of a glioblastoma cell
line to temozolimide when TROY expression is suppressed.
[0032] FIG. 20 depicts reduced sensitivity of a glioblastoma cell
line to temozolimide when the cell line is transfected with an
HA-tagged TROY construct.
[0033] FIG. 21 depicts reduced migration of a glioblastoma cell
line when Dock180 and Dock7 expression are suppressed.
[0034] FIG. 22 depicts reduced depth of invasion of glioblastoma
cells when Dock7 expression is suppressed using two different
siRNAs.
[0035] FIG. 23 depicts a Western blot showing TROY expression in
glioblastoma xenografts grown in murine brain.
[0036] Elements and acts in the figures are illustrated for
simplicity and have not necessarily been rendered according to any
particular sequence or embodiment.
DETAILED DESCRIPTION OF THE INVENTION
[0037] In the following description, and for the purposes of
explanation, numerous specific details are set forth in order to
provide a thorough understanding of the various aspects of the
invention. It will be understood, however, by those skilled in the
relevant arts, that the present invention may be practiced without
these specific details. In other instances, known structures and
devices are shown or discussed more generally in order to avoid
obscuring the invention.
[0038] Gliomas, primary brain tumors that derive from glial support
cells, are the most common primary tumor of the adult central
nervous system and will result in an estimated 13,000 deaths in
2010 (See References 1, 3, 11, and 12). Adult gliomas of astrocytic
origin (astrocytomas) comprise a spectrum of neoplasms that are
generally classified by WHO standards into low-grade benign tumors
(i.e. juvenile pilocytic astrocytoma, diffuse astrocytoma) and
high-grade malignant tumors (i.e. anaplastic astrocytoma and
glioblastoma multiforme; GBM). Patients diagnosed with grade IV
GBM, the most aggressive malignant glioma, have a median survival
of 9-12 months after the onset of clinical symptoms (See References
11-13). Molecular analyses of glioma specimens have identified
several common genetic alterations (e.g., p16INK4a deletion) and
gene expression changes (e.g., EGFR overexpression) that may
contribute to glioblastoma formation (See References 14 and
15).
[0039] In general, gliomas are extremely difficult to treat using
conventional approaches (See References 12-16.) This is primarily
due to the intrinsic propensity of glioma cells to exit the tumor
core and invade the adjacent normal brain parenchyma (See
References 3 and 4). These migrating cells escape surgical
resection and are poorly targeted by radiation or chemotherapy.
They sometimes travel over long distances, frequently along blood
vessel and fiber tracts, and then initiate secondary tumor growth
at their final destination. This distinguishing invasive ability is
not shared by nonglial cells that metastasize from other primary
tumor sites (e.g. breast) to brain tissue. The invasion of glioma
cells is likely triggered by a presently undefined signal or
signals that promote a cascade of cellular responses, including
cell elongation, integrin-mediated cell attachment to extracellular
matrix (ECM) molecules, the production and secretion of
ECM-degrading enzymes, and cell movement (See References 17 and
18).
[0040] Migrating glioma cells exhibit decreased susceptibility to
pro-apoptotic agents (See Reference 19) providing them with an
additional mechanism for resisting current radiological and
chemotherapeutic treatment modalities.
[0041] TROY (TNFRSF19) is an orphan member of the TNFR superfamily
that is highly expressed in embryonic and adult CNS, and developing
hair follicles (See References 5-10). During mouse embryogenesis,
TROY mRNA is detected in many developing tissues including the limb
buds, eyelids, whiskers, mammary glands, epidermis, bronchial,
tongue, dental and gastric epithelium as well as the germinal zones
of the CNS including the ventricular zone and subventricular zone.
However, in adult animals, TROY expression changes and is primarily
restricted to hair follicles and neuron-like cells in the cerebrum,
cerebral cortex, developing olfactory system as well as dorsal root
and retinal ganglion neurons (See References 5-10) In the
peripheral nervous system, TROY functions as a co-receptor for the
ligand-binding Nogo-66 receptor 1 (NgR1) to form the
TROY/NgR1/LINGO complex that activates the RhoA pathway to inhibit
neurite outgrowth of dorsal root ganglion neurons in adult mice
(See References 6 and 9). In humans, TROY mRNA is primarily
expressed in the brain and also the prostate, whereas low or
undetectable levels are observed in the heart, lung, liver, thymus,
uterus, skeletal muscle, spleen, colon testis, kidney and
peripheral blood lymphocytes (See Reference 20). The reason or
mechanism for this "switch-off" of TROY expression after birth is
unclear, but its strict control indicates that aberrant expression
may be detrimental. Indeed, it has been recently reported that TROY
is highly expressed in primary and metastatic melanoma cells, but
not in melanocytes found in normal skin biopsies and primary skin
cell cultures (See Reference 21).
[0042] Herein, the Inventor demonstrates that TROY serves as a
target or marker of invasive glioblastoma, that its expression is
linked to poor therapeutic outcome and that it serves as a marker
of resistance to temozolimide and as a marker of sensitivity to
classes of drugs that treat glioblastoma by targeting pathways that
contribute to glioma cell migration and invasion.
[0043] A marker may be any molecular structure produced by a cell,
expressed inside the cell, accessible on the cell surface, or
secreted by the cell. A marker may be any protein, carbohydrate,
fat, nucleic acid, catalytic site, or any combination of these such
as an enzyme, glycoprotein, cell membrane, virus, cell, organ,
organelle, or any uni- or multimolecular structure or any other
such structure now known or yet to be disclosed whether alone or in
combination. A marker may also be called a target and the terms are
used interchangeably.
[0044] A marker may be represented by the sequence of a nucleic
acid from which it can be derived. Examples of such nucleic acids
include miRNA, tRNA, siRNA, mRNA, cDNA, or genomic DNA sequences.
While a marker may be represented by the sequence of a single
nucleic acid strand (e.g. 5'.fwdarw.3'), nucleic acid reagents that
bind the marker may also bind to the complementary strand (e.g.
3'.fwdarw.5'). Alternatively, a marker may be represented by a
protein sequence. The concept of a marker is not limited to the
products of the exact nucleic acid sequence or protein sequence by
which it may be represented. Rather, a marker encompasses all
molecules that may be detected by a method of assessing the
expression of the marker.
[0045] Examples of molecules encompassed by a marker include point
mutations, silent mutations, deletions, frameshift mutations,
translocations, alternative splicing derivatives, differentially
methylated sequences, differentially modified protein sequences,
truncations, soluble forms of cell membrane associated markers, and
any other variation that results in a product that may be
identified as the marker. The following nonlimiting examples are
included for the purposes of clarifying this concept: If expression
of a specific marker in a sample is assessed by RTPCR, and if the
sample expresses an mRNA sequence different from the sequence used
to identify the specific marker by one or more nucleotides, but the
marker may still be detected using RTPCR, then the specific marker
encompasses the sequence present in the sample. Alternatively if
expression of a specific marker in a sample is assessed by an
antibody and the amino acid sequence of the marker in the sample
differs from a sequence used to identify marker by one or more
amino acids, but the antibody is still able to bind to the version
of the marker in the sample, then the specific marker encompasses
the sequence present in the sample.
[0046] Expression encompasses any and all processes through which
material derived from a nucleic acid template may be produced.
Expression thus includes processes such as RNA transcription, mRNA
splicing, protein translation, protein folding, post-translational
modification, membrane transport, associations with other
molecules, addition of carbohydrate moieties to proteins,
phosphorylation, protein complex formation and any other process
along a continuum that results in biological material derived from
genetic material whether in vitro, in vivo, or ex vivo. Expression
also encompasses all processes through which the production of
material derived from a nucleic acid template may be actively or
passively suppressed. Such processes include all aspects of
transcriptional and translational regulation. Examples include
heterochromatic silencing, differential methylation, transcription
factor inhibition, any form of RNAi silencing, microRNA silencing,
alternative splicing, protease digestion, posttranslational
modification, and alternative protein folding.
[0047] Expression may be assessed by any number of methods used to
detect material derived from a nucleic acid template used currently
in the art and yet to be developed. Examples of such methods
include any nucleic acid detection method including the following
nonlimiting examples, microarray analysis, RNA in situ
hybridization, RNAse protection assay, Northern blot, reverse
transcriptase PCR, quantitative PCR, quantitative reverse
transcriptase PCR, quantitative real-time reverse transcriptase
PCR, reverse transcriptase treatment followed by direct sequencing,
direct sequencing of genomic DNA, or any other method of detecting
a specific nucleic acid now known or yet to be disclosed. Other
examples include any process of assessing protein expression
including flow cytometry, immunohistochemistry, ELISA, Western
blot, and immunoaffinity chromatograpy, HPLC, mass spectrometry,
protein microarray analysis, PAGE analysis, isoelectric focusing,
2-D gel electrophoresis, or any enzymatic assay. Methods of
detecting expression may include methods of purifying nucleic acid,
protein, or some other material depending on the type of marker.
Any method of nucleic acid purification may be used, depending on
the type of marker. Examples include phenol alcohol extraction,
ethanol extraction, guanidium isothionate extraction, gel
purification, size exclusion chromatography, cesium chloride
preparations, and silica resin preparation. Any method of protein
purification may be used, also depending on the type of marker.
Examples include size exclusion chromatography, hydrophobic
interaction chromatography, ion exchange chromatography, affinity
chromatograpy (including affinity chromatography of tagged
proteins), metal binding, immunoaffinity chromatography, and
HPLC.
[0048] Nucleic acid amplification is a process by which copies of a
nucleic acid may be made from a source nucleic acid. Nucleic acids
that may be subjected to amplification may be from any source. In
some nucleic amplification methods, the copies are generated
exponentially. Examples of nucleic acid amplification include but
are not limited to: the polymerase chain reaction (PCR), ligase
chain reaction (LCR), self-sustained sequence replication (3SR),
nucleic acid sequence based amplification (NASBA), strand
displacement amplification (SDA), amplification with Q.beta.
replicase, whole genome amplification with enzymes such as .phi.29,
whole genome PCR, in vitro transcription with any RNA polymerase,
or any other method by which copies of a desired sequence are
generated.
[0049] Polymerase chain reaction (PCR) is a particular method of
amplifying DNA, generally involving the mixing of a nucleic sample,
two or more primers, a DNA polymerase, which may be a thermostable
DNA polymerase such as Taq or Pfu, and deoxyribose nucleoside
triphosphates (dNTP's). In general, the reaction mixture is
subjected to temperature cycles comprising a denaturation stage,
(typically 80-100.degree. C.) an annealing stage with a temperature
that is selected based on the melting temperature (Tm) of the
primers and the degeneracy of the primers, and an extension stage
(for example 40-75.degree. C.) In real-time PCR analysis,
additional reagents, methods, optical detection systems, and
devices are used that allow a measurement of the magnitude of
fluorescence in proportion to concentration of amplified DNA. In
such analyses, incorporation of fluorescent dye into the amplified
strands may be detected or labeled probes that bind to a specific
sequence during the annealing phase release their fluorescent tags
during the extension phase. Either of these will allow a
quantification of the amount of specific DNA present in the initial
sample. Often, the result of a real-time PCR will be expressed in
the terms of cycle threshold (Ct) values. The Ct represents the
number of PCR cycles for the fluorescent signal from a real-time
PCR reaction to cross a threshold value of fluorescence. Ct is
inversely proportional to the amount of target nucleic acid
originally present in the sample. RNA may be detected by PCR
analysis by creating a DNA template from RNA through a reverse
transcriptase enzyme.
[0050] Other methods used to assess expression include the use of
natural or artificial ligands capable of specifically binding a
marker. Such ligands include antibodies, antibody complexes,
conjugates, natural ligands, small molecules, nanoparticles, or any
other molecular entity capable of specific binding to a marker.
Antibodies may be monoclonal, polyclonal, or any antibody fragment
including an Fab, F(ab).sub.2, Fv, scFv, phage display antibody,
peptibody, multispecific ligand, or any other reagent with specific
binding to a marker. Ligands may be associated with a label such as
a radioactive isotope or chelate thereof, dye (fluorescent or
nonfluorescent), stain, enzyme, metal, or any other substance
capable of aiding a machine or a human eye from differentiating a
cell expressing a marker from a cell not expressing a marker.
Additionally, expression may be assessed by monomeric or multimeric
ligands associated with substances capable of killing the cell.
Such substances include protein or small molecule toxins,
cytokines, pro-apoptotic substances, pore forming substances,
radioactive isotopes, or any other substance capable of killing a
cell.
[0051] Differential expression encompasses any detectable
difference between the expression of a marker in one sample
relative to the expression of the marker in another sample.
Differential expression may be assessed by a detector, an
instrument containing a detector, or by aided or unaided human eye.
Examples include but are not limited to differential staining of
cells in an IHC assay configured to detect a marker, differential
detection of bound RNA on a microarray to which a sequence capable
of binding to the marker is bound, differential results in
measuring RTPCR measured in the number of PCR cycles necessary to
reach a particular optical density at a wavelength at which a
double stranded DNA binding dye (e.g. SYBR Green) incorporates,
differential results in measuring label from a reporter probe used
in a real-time RTPCR reaction, differential detection of
fluorescence on cells using a flow cytometer, differential
intensities of bands in a Northern blot, differential intensities
of bands in an RNAse protection assay, differential cell death
measured by apoptotic markers, differential cell death measured by
shrinkage of a tumor, or any method that allows a detection of a
difference in signal between one sample or set of samples and
another sample or set of samples.
[0052] The expression of the marker in a sample may be compared to
a level of expression predetermined to predict the presence or
absence of a particular physiological characteristic. The level of
expression may be derived from a single control or a set of
controls. A control may be any sample with a previously determined
level of expression. A control may comprise material within the
sample or material from sources other than the sample.
Alternatively, the expression of a marker in a sample may be
compared to a control that has a level of expression predetermined
to signal or not signal a cellular or physiological characteristic.
This level of expression may be derived from a single source of
material including the sample itself or from a set of sources.
Comparison of the expression of the marker in the sample to a
particular level of expression results in a prediction that the
sample exhibits or does not exhibit the cellular or physiological
characteristic.
[0053] Prediction of a cellular or physiological characteristic
includes the prediction of any cellular or physiological state that
may be predicted by assessing the expression of a marker. Examples
include the identity of a cell as a particular cell including a
particular normal or cancer cell type, the likelihood that one or
more diseases is present or absent, the likelihood that a present
disease will progress, remain unchanged, or regress, the likelihood
that a disease will respond or not respond to a particular therapy,
or any other outcome. Further examples include the likelihood that
a cell will move, senesce, apoptose, differentiate, metastasize, or
change from any state to any other state or maintain its current
state.
[0054] Expression of a marker in a sample may be more or less than
that of a level predetermined to predict the presence or absence of
a cellular or physiological characteristic. The expression of the
marker in the sample may be more than 1,000,000.times.,
100,000.times., 10,000.times., 1000.times., 100.times., 10.times.,
5.times., 2.times., 1.times., 0.5.times., 0.1.times., 0.01.times.,
0.001.times., 0.0001.times., 0.00001.times., 0.000001.times.,
0.0000001.times. or less than that of a level predetermined to
predict the presence or absence of a cellular or physiological
characteristic.
[0055] The invention contemplates assessing the expression of the
marker in any biological sample from which the expression may be
assessed. One skilled in the art would know to select a particular
biological sample and how to collect said sample depending upon the
marker that is being assessed. Examples of sources of samples
include but are not limited to biopsy or other in vivo or ex vivo
analysis of prostate, breast, skin, muscle, facia, brain,
endometrium, lung, head and neck, pancreas, small intestine, blood,
liver, testes, ovaries, colon, skin, stomach, esophagus, spleen,
lymph node, bone marrow, kidney, placenta, or fetus. In some
aspects of the invention, the sample comprises a fluid sample, such
as peripheral blood, lymph fluid, ascites, serous fluid, pleural
effusion, sputum, cerebrospinal fluid, amniotic fluid, lacrimal
fluid, stool, or urine. Samples include single cells, whole organs
or any fraction of a whole organ, in any condition including in
vitro, ex vivo, in vivo, post-mortem, fresh, fixed, or frozen.
[0056] One type of cellular or physiological characteristic is the
risk that a particular disease outcome will occur. Assessing this
risk includes the performing of any type of test, assay,
examination, result, readout, or interpretation that correlates
with an increased or decreased probability that an individual has
had, currently has, or will develop a particular disease, disorder,
symptom, syndrome, or any condition related to health or bodily
state. Examples of disease outcomes include, but need not be
limited to survival, death, progression of existing disease,
remission of existing disease, initiation of onset of a disease in
an otherwise disease-free subject, or the continued lack of disease
in a subject in which there has been a remission of disease.
Assessing the risk of a particular disease encompasses diagnosis in
which the type of disease afflicting a subject is determined.
Assessing the risk of a disease outcome also encompasses the
concept of prognosis. A prognosis may be any assessment of the risk
of disease outcome in an individual in which a particular disease
has been diagnosed. Assessing the risk further encompasses
prediction of therapeutic response in which a treatment regimen is
chosen based on the assessment. Assessing the risk also encompasses
a prediction of overall survival after diagnosis.
[0057] Determining the level of expression that signifies a
physiological or cellular characteristic may be assessed by any of
a number of methods. The skilled artisan will understand that
numerous methods may be used to select a level of expression for a
particular marker or a plurality of markers that signifies a
particular physiological or cellular characteristics. In diagnosing
the presence of a disease, a threshold value may be obtained by
performing the assay method on samples obtained from a population
of patients having a certain type of disease (cancer for example),
and from a second population of subjects that do not have the
disease. In assessing disease outcome or the effect of treatment, a
population of patients, all of which have, a disease such as
cancer, may be followed for a period of time. After the period of
time expires, the population may be divided into two or more
groups. For example, the population may be divided into a first
group of patients whose disease progresses to a particular endpoint
and a second group of patients whose disease does not progress to
the particular endpoint. Examples of endpoints include disease
recurrence, death, metastasis or other states to which disease may
progress. If expression of the marker in a sample is more similar
to the predetermined expression of the marker in one group relative
to the other group, the sample may be assigned a risk of having the
same outcome as the patient group to which it is more similar.
[0058] In addition, one or more levels of expression of the marker
may be selected that signify a particular physiological or cellular
characteristic. For example, Receiver Operating Characteristic
curves, or "ROC" curves, may be calculated by plotting the value of
a variable versus its relative frequency in two populations. For
any particular marker, a distribution of marker expression levels
for subjects with and without a disease may overlap. This indicates
that the test does not absolutely distinguish between the two
populations with complete accuracy. The area of overlap indicates
where the test cannot distinguish the two groups. A threshold is
selected. Expression of the marker in the sample above the
threshold indicates the sample is similar to one group and
expression of the marker below the threshold indicates the sample
is similar to the other group. The area under the ROC curve is a
measure of the probability that the expression correctly indicated
the similarity of the sample to the proper group. See, e.g., Hanley
et al., Radiology 143: 29-36 (1982) hereby incorporated by
reference.
[0059] Additionally, levels of expression may be established by
assessing the expression of a marker in a sample from one patient,
assessing the expression of additional samples from the same
patient obtained later in time, and comparing the expression of the
marker from the later samples with the initial sample or samples.
This method may be used in the case of markers that indicate, for
example, progression or worsening of disease or lack of efficacy of
a treatment regimen or remission of a disease or efficacy of a
treatment regimen.
[0060] Other methods may be used to assess how accurately the
expression of a marker signifies a particular physiological or
cellular characteristic. Such methods include a positive likelihood
ratio, negative likelihood ratio, odds ratio, and/or hazard ratio.
In the case of a likelihood ratio, the likelihood that the
expression of the marker would be found in a sample with a
particular cellular or physiological characteristic is compared
with the likelihood that the expression of the marker would be
found in a sample lacking the particular cellular or physiological
characteristic.
[0061] An odds ratio measures effect size and describes the amount
of association or non-independence between two groups. An odds
ratio is the ratio of the odds of a marker being expressed in one
set of samples versus the odds of the marker being expressed in the
other set of samples. An odds ratio of 1 indicates that the event
or condition is equally likely to occur in both groups. An odds
ratio greater or less than 1 indicates that expression of the
marker is more likely to occur in one group or the other depending
on how the odds ratio calculation was set up. A hazard ratio may be
calculated by estimate of relative risk. Relative risk is the
chance that a particular event will take place. It is a ratio of
the probability that an event such as development or progression of
a disease will occur in samples that exceed a threshold level of
expression of a marker over the probability that the event will
occur in samples that do not exceed a threshold level of expression
of a marker. Alternatively, a hazard ratio may be calculated by the
limit of the number of events per unit time divided by the number
at risk as the time interval decreases. In the case of a hazard
ratio, a value of 1 indicates that the relative risk is equal in
both the first and second groups. A value greater or less than 1
indicates that the risk is greater in one group or another,
depending on the inputs into the calculation.
[0062] Additionally, multiple threshold levels of expression may be
determined. This can be the case in so-called "tertile,"
"quartile," or "quintile" analyses. In these methods, multiple
groups can be considered together as a single population, and are
divided into 3 or more bins having equal numbers of individuals.
The boundary between two of these "bins" may be considered
threshold levels of expression indicating a particular level of
risk of a disease developing or signifying a physiological or
cellular state. A risk may be assigned based on which "bin" a test
subject falls into.
[0063] A subject includes any human or non-human mammal, including
for example: a primate, cow, horse, pig, sheep, goat, dog, cat, or
rodent, capable of developing cancer including human patients that
are suspected of having cancer, that have been diagnosed with
cancer, or that have a family history of cancer. Methods of
identifying subjects suspected of having cancer include but are not
limited to: physical examination, family medical history, subject
medical history including exposure to environmental factors,
biopsy, or any of a number of imaging technologies such as
ultrasonography, computed tomography, magnetic resonance imaging,
magnetic resonance spectroscopy, or positron emission
tomography.
[0064] Cancer cells include any cells derived from a tumor,
neoplasm, cancer, precancer, cell line, malignancy, or any other
source of cells that have the potential to expand and grow to an
unlimited degree. Cancer cells may be derived from naturally
occurring sources or may be artificially created. Cancer cells may
also be capable of invasion into other tissues and metastasis.
Cancer cells further encompass any malignant cells that have
invaded other tissues and/or metastasized. One or more cancer cells
in the context of an organism may also be called a cancer, tumor,
neoplasm, growth, malignancy, or any other term used in the art to
describe cells in a cancerous state.
[0065] Examples of cancers that could serve as sources of cancer
cells include solid tumors such as fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,
angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal
cancer, kidney cancer, pancreatic cancer, bone cancer, breast
cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach
cancer, oral cancer, nasal cancer, throat cancer, squamous cell
carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland
carcinoma, sebaceous gland carcinoma, papillary carcinoma,
papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,
bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct
carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms'
tumor, cervical cancer, uterine cancer, testicular cancer, small
cell lung carcinoma, bladder carcinoma, lung cancer, epithelial
carcinoma, glioma, glioblastoma multiforme, astrocytoma,
medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,
skin cancer, melanoma, neuroblastoma, and retinoblastoma.
[0066] Additional cancers that may serve as sources of cancer cells
include blood borne cancers such as acute lymphoblastic leukemia
("ALL,"), acute lymphoblastic B-cell leukemia, acute lymphoblastic
T-cell leukemia, acute myeloblastic leukemia ("AML"), acute
promyelocytic leukemia ("APL"), acute monoblastic leukemia, acute
erythroleukemic leukemia, acute megakaryoblastic leukemia, acute
myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute
undifferentiated leukemia, chronic myelocytic leukemia ("CML"),
chronic lymphocytic leukemia ("CLL"), hairy cell leukemia, multiple
myeloma, lymphoblastic leukemia, myelogenous leukemia, lymphocytic
leukemia, myelocytic leukemia, Hodgkin's disease, non-Hodgkin's
Lymphoma, Waldenstrom's macroglobulinemia, Heavy chain disease, and
Polycythemia vera.
[0067] The present invention further provides kits to be used in
assessing the expression of a particular RNA in a sample from a
subject to assess the risk of developing disease. Kits include any
combination of components that facilitates the performance of an
assay. A kit that facilitates assessing the expression of an RNA
may include suitable nucleic acid-based and immunological reagents
as well as suitable buffers, control reagents, and printed
protocols.
[0068] Kits that facilitate nucleic acid based methods may further
include one or more of the following: specific nucleic acids such
as oligonucleotides, labeling reagents, enzymes including PCR
amplification reagents such as Taq or Pfu; reverse transcriptase,
or one or more other polymerases, and/or reagents that facilitate
hybridization. Specific nucleic acids may include nucleic acids,
polynucleotides, oligonucleotides (DNA, or RNA), or any combination
of molecules that includes one or more of the above, or any other
molecular entity capable of specific binding to a nucleic acid
marker. In one aspect of the invention, the specific nucleic acid
comprises one or more oligonucleotides capable of hybridizing to
the marker.
[0069] A specific nucleic acid may include a label. A label may be
any substance capable of aiding a machine, detector, sensor,
device, or enhanced or unenhanced human eye from differentiating a
sample that that displays positive expression from a sample that
displays reduced expression. Examples of labels include but are not
limited to: a radioactive isotope or chelate thereof, a dye
(fluorescent or nonfluorescent), stain, enzyme, or nonradioactive
metal. Specific examples include but are not limited to:
fluorescein, biotin, digoxigenin, alkaline phosphatase, biotin,
streptavidin, .sup.3H, .sup.14C, .sup.32P, .sup.35S, or any other
compound capable of emitting radiation, rhodamine,
4-(4'-dimethylaminophenylazo) benzoic acid ("Dabcyl");
4-(4'-dimethylamino-phenylazo)sulfonic acid (sulfonyl chloride)
("Dabsyl"); 5-((2-aminoethyl)-amino)-naphtalene-1-sulfonic acid
("EDANS"); Psoralene derivatives, haptens, cyanines, acridines,
fluorescent rhodol derivatives, cholesterol derivatives;
ethylenediaminetetraaceticacid ("EDTA") and derivatives thereof or
any other compound that signals the presence of the labeled nucleic
acid. In one embodiment of the invention, the label includes one or
more dyes optimized for use in genotyping. Examples of such dyes
include but are not limited to: dR110, 5-FAM, 6FAM, dR6G, JOE, HEX,
VIC, TET, dTAMRA, TAMRA, NED, dROX, PET, and LIZ.
[0070] An oligonucleotide is a reagent capable of binding a nucleic
acid sequence. An oligonucleotide may be any polynucleotide of at
least 2 nucleotides. Oligonucleotides may be less than 10, less
than 15, less than 20, less than 30, less than 40, less than 50,
less than 75, less than 100, less than 200, less than 500, or more
than 500 nucleotides in length. While oligonucleotides are often
linear, they may, depending on their sequence and conditions,
assume a two- or three-dimensional structure. Oligonucleotides may
be chemically synthesized by any of a number of methods including
sequential synthesis, solid phase synthesis, or any other synthesis
method now known or yet to be disclosed. Alternatively,
oligonucleotides may be produced by recombinant DNA based methods.
One skilled in the art would understand the length of
oligonucleotide necessary to perform a particular task.
Oligonucleotides may be directly labeled, used as primers in PCR or
sequencing reactions, or bound directly to a solid substrate as in
oligonucleotide arrays.
[0071] A nucleotide is an individual deoxyribonucleotide or
ribonucleotide base. Examples of nucleotides include but are not
limited to: adenine, thymine, guanine, cytosine, and uracil, which
may be abbreviated as A, T, G, C, or U in representations of
oligonucleotide or polynucleotide sequence. Any molecule of two or
more nucleotide bases, whether DNA or RNA, may be termed a nucleic
acid.
[0072] An oligonucleotide used to detect to an allele may be
affixed to a solid substrate. Alternatively, the sample may be
affixed to a solid substrate and the nucleic acid reagent placed
into a mixture. For example, the nucleic acid reagent may be bound
to a substrate in the case of an array or the sample may be bound
to a substrate as the case of a Southern Blot, Northern blot or
other method that affixes the sample to a substrate. A nucleic acid
reagent or sample may be covalently bound to the substrate or it
may be bound by some non covalent interaction including
electrostatic, hydrophobic, hydrogen bonding, Van Der Waals,
magnetic, or any other interaction by which an oligonucleotide may
be attached to a substrate while maintaining its ability to
recognize the allele to which it has specificity. A substrate may
be any solid or semi solid material onto which a probe may be
affixed, attached or printed, either singly or in the formation of
a microarray. Examples of substrate materials include but are not
limited to polyvinyl, polysterene, polypropylene, polyester or any
other plastic, glass, silicon dioxide or other silanes, hydrogels,
gold, platinum, microbeads, micelles and other lipid formations,
nitrocellulose, or nylon membranes. The substrate may take any
shape, including a spherical bead or flat surface.
[0073] A nucleotide is an individual deoxyribonucleotide or
ribonucleotide base. Examples of nucleotides include but are not
limited to: adenine, thymine, guanine, cytosine, and uracil, which
may be abbreviated as A, T, G, C, or U in representations of
oligonucleotide or polynucleotide sequence.
[0074] In some aspects of the invention, the probe may be affixed
to a solid substrate. In other aspects of the invention, the sample
may be affixed to a solid substrate. A probe or sample may be
covalently bound to the substrate or it may be bound by some non
covalent interaction including electrostatic, hydrophobic, hydrogen
bonding, Van Der Waals, magnetic, or any other interaction by which
a probe such as an oligonucleotide probe may be attached to a
substrate while maintaining its ability to recognize the allele to
which it has specificity. A substrate may be any solid or semi
solid material onto which a probe may be affixed, attached or
printed, either singly or in the formation of a microarray.
Examples of substrate materials include but are not limited to
polyvinyl, polysterene, polypropylene, polyester or any other
plastic, glass, silicon dioxide or other silanes, hydrogels, gold,
platinum, microbeads, micelles and other lipid formations,
nitrocellulose, or nylon membranes. The substrate may take any
form, including a spherical bead or flat surface. For example, the
probe may be bound to a substrate in the case of an array. The
sample may be bound to a substrate as (for example) the case of a
Southern Blot, Northern blot or other method that affixes the
sample to a substrate.
[0075] Kits may also contain reagents that detect proteins, often
through the use of an antibody. These kits will contain one or more
specific antibodies, buffers, and other reagents configured to
detect binding of the antibody to the specific epitope. One or more
of the antibodies may be labeled with a fluorescent, enzymatic,
magnetic, metallic, chemical, or other label that signifies and/or
locates the presence of specifically bound antibody. The kit may
also contain one or more secondary antibodies that specifically
recognize epitopes on other antibodies. These secondary antibodies
may also be labeled. The concept of a secondary antibody also
encompasses non-antibody ligands that specifically bind an epitope
or label of another antibody. For example, streptavidin or avidin
may bind to biotin conjugated to another antibody. Such a kit may
also contain enzymatic substrates that change color or some other
property in the presence of an enzyme that is conjugated to one or
more antibodies included in the kit.
[0076] A kit may also contain an indication of a result of the use
of the kit that signifies a particular physiological or cellular
characteristic. An indication includes any guide to a result that
would signal the presence or absence of any physiological or
cellular state that the kit is configured to predict. For example,
the indication may be expressed numerically, expressed as a color
or density of a color, expressed as an intensity of a band, derived
from a standard curve, or expressed in comparison to a control. The
indication may be communicated through the use of a writing that
may be contained physically in or on the kit (on a piece of paper
for example), posted on the Internet, mailed to the user separately
from the kit, or embedded in a software package. The writing may be
in any medium that communicates how the result may be used to
predict the cellular or physiological characteristic such as a
printed document, a photograph, sound, color, or any combination
thereof.
[0077] The invention further encompasses pharmaceutical
compositions that include the disclosed compound as an ingredient.
Such pharmaceutical compositions may take any physical form
necessary depending on a number of factors including the desired
method of administration and the physicochemical and stereochemical
form taken by the disclosed compound or pharmaceutically acceptable
salts of the compound. Such physical forms include a solid, liquid,
gas, sol, gel, aerosol, or any other physical form now known or yet
to be disclosed. The concept of a pharmaceutical composition
including the disclosed compound also encompasses the disclosed
compound or a pharmaceutically acceptable salt thereof without any
other additive. The physical form of the invention may affect the
route of administration and one skilled in the art would know to
choose a route of administration that takes into consideration both
the physical form of the compound and the disorder to be treated.
Pharmaceutical compositions that include the disclosed compound may
be prepared using methodology well known in the pharmaceutical art.
A pharmaceutical composition that includes the disclosed compound
may include a second effective compound of a distinct chemical
formula from the disclosed compound. This second effective compound
may have the same or a similar molecular target as the target or it
may act upstream or downstream of the molecular target of the
disclosed compound with regard to one or more biochemical
pathways.
[0078] Pharmaceutical compositions including the disclosed compound
include materials capable of modifying the physical form of a
dosage unit. In one nonlimiting example, the composition includes a
material that forms a coating that holds in the compound. Materials
that may be used in such a coating, include, for example, sugar,
shellac, gelatin, or any other inert coating agent.
[0079] Pharmaceutical compositions including the disclosed compound
may be prepared as a gas or aerosol. Aerosols encompass a variety
of systems including colloids and pressurized packages. Delivery of
a composition in this form may include propulsion of a
pharmaceutical composition including the disclosed compound through
use of liquefied gas or other compressed gas or by a suitable pump
system. Aerosols may be delivered in single phase, bi-phasic, or
tri-phasic systems.
[0080] In some aspects of the invention, the pharmaceutical
composition including the disclosed compound is in the form of a
solvate. Such solvates are produced by the dissolution of the
disclosed compound in a pharmaceutically acceptable solvent.
Pharmaceutically acceptable solvents include any mixtures of more
than one solvent. Such solvents may include pyridine, chloroform,
propan-1-ol, ethyl oleate, ethyl lactate, ethylene oxide, water,
ethanol, and any other solvent that delivers a sufficient quantity
of the disclosed compound to treat the affliction without serious
complications arising from the use of the solvent in a majority of
patients.
[0081] Pharmaceutical compositions that include the disclosed
compound may also include a pharmaceutically acceptable carrier.
Carriers include any substance that may be administered with the
disclosed compound with the intended purpose of facilitating,
assisting, or helping the administration or other delivery of the
compound. Carriers include any liquid, solid, semisolid, gel,
aerosol or anything else that may be combined with the disclosed
compound to aid in its administration. Examples include diluents,
adjuvants, excipients, water, oils (including petroleum, animal,
vegetable or synthetic oils.) Such carriers include particulates
such as a tablet or powder, liquids such as an oral syrup or
injectable liquid, and inhalable aerosols. Further examples include
saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal
silica, and urea. Such carriers may further include binders such as
ethyl cellulose, carboxymethylcellulose, microcrystalline
cellulose, or gelatin; excipients such as starch, lactose or
dextrins; disintegrating agents such as alginic acid, sodium
alginate, Primogel, and corn starch; lubricants such as magnesium
stearate or Sterotex; glidants such as colloidal silicon dioxide;
sweetening agents such as sucrose or saccharin, a flavoring agent
such as peppermint, methyl salicylate or orange flavoring, or
coloring agents. Further examples of carriers include polyethylene
glycol, cyclodextrin, oils, or any other similar liquid carrier
that may be formulated into a capsule. Still further examples of
carriers include sterile diluents such as water for injection,
saline solution, physiological saline, Ringer's solution, isotonic
sodium chloride, fixed oils such as synthetic mono or digylcerides,
polyethylene glycols, glycerin, cyclodextrin, propylene glycol or
other solvents; antibacterial agents such as benzyl alcohol or
methyl paraben; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose,
thickening agents, lubricating agents, and coloring agents.
[0082] The pharmaceutical composition including the disclosed
compound may take any of a number of formulations depending on the
physicochemical form of the composition and the type of
administration. Such forms include solutions, suspensions,
emulsions, tablets, pills, pellets, capsules, capsules including
liquids, powders, sustained-release formulations, directed release
formulations, lyophylates, suppositories, emulsions, aerosols,
sprays, granules, powders, syrups, elixirs, or any other
formulation now known or yet to be disclosed. Additional examples
of suitable pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin, hereby incorporated by
reference in its entirety.
[0083] Methods of administration include, but are not limited to,
oral administration and parenteral administration. Parenteral
administration includes, but is not limited to intradermal,
intramuscular, intraperitoneal, intravenous, subcutaneous,
intranasal, epidural, sublingual, intramsal, intracerebral,
intraventricular, intrathecal, intravaginal, transdermal, rectal,
by inhalation, or topically to the ears, nose, eyes, or skin. Other
methods of administration include but are not limited to infusion
techniques including infusion or bolus injection, by absorption
through epithelial or mucocutaneous linings such as oral mucosa,
rectal and intestinal mucosa. Compositions for parenteral
administration may be enclosed in ampoule, a disposable syringe or
a multiple-dose vial made of glass, plastic or other material.
[0084] Administration may be systemic or local. Local
administration is administration of the disclosed compound to the
area in need of treatment. Examples include local infusion during
surgery; topical application, by local injection; by a catheter; by
a suppository; or by an implant. Administration may be by direct
injection at the site (or former site) of a cancer, tumor, or
precancerous tissue or into the central nervous system by any
suitable route, including intraventricular and intrathecal
injection. Intraventricular injection can be facilitated by an
intraventricular catheter, for example, attached to a reservoir,
such as an Ommaya reservoir. Pulmonary administration may be
achieved by any of a number of methods known in the art. Examples
include use of an inhaler or nebulizer, formulation with an
aerosolizing agent, or via perfusion in a fluorocarbon or synthetic
pulmonary surfactant. The disclosed compound may be delivered in
the context of a vesicle such as a liposome or any other natural or
synthetic vesicle.
[0085] A pharmaceutical composition formulated so as to be
administered by injection may be prepared by dissolving the
disclosed compound with water so as to form a solution. In
addition, a surfactant may be added to facilitate the formation of
a homogeneous solution or suspension. Surfactants include any
complex capable of non-covalent interaction with the disclosed
compound so as to facilitate dissolution or homogeneous suspension
of the compound.
[0086] Pharmaceutical compositions including the disclosed compound
may be prepared in a form that facilitates topical or transdermal
administration. Such preparations may be in the form of a liquid
solution, cream, paste, lotion, shake lotion, powder, emulsion,
ointment, gel base, transdermal patch or iontophoresis device.
Examples of bases used in such compositions include opetrolatum,
lanolin, polyethylene glycols, beeswax, mineral oil, diluents such
as water and alcohol, and emulsifiers and stabilizers, thickening
agents, or any other suitable base now known or yet to be
disclosed.
[0087] Addition of a pharmaceutical composition to cancer cells
includes all actions by which an effect of the pharmaceutical
composition on the cancer cell is realized. The type of addition
chosen will depend upon whether the cancer cells are in vivo, ex
vivo, or in vitro, the physical or chemical properties of the
pharmaceutical composition, and the effect the composition is to
have on the cancer cell. Nonlimiting examples of addition include
addition of a solution including the pharmaceutical composition to
tissue culture media in which in vitro cancer cells are growing;
any method by which a pharmaceutical composition may be
administered to an animal including intravenous, per os,
parenteral, or any other of the methods of administration; or the
activation or inhibition of cells that in turn have effects on the
cancer cells such as immune cells (e.g. macophages and CD8+ T
cells) or endothelial cells that may differentiate into blood
vessel structures in the process of angiogenesis or
vasculogenesis.
[0088] Determination of an effective amount of the disclosed
compound is within the capability of those skilled in the art,
especially in light of the detailed disclosure provided herein. The
effective amount of a pharmaceutical composition used to effect a
particular purpose as well as a pharmacologically acceptable dose
determined by toxicity, excretion, and overall tolerance may be
determined in cell cultures or experimental animals by
pharmaceutical and toxicological procedures either known now by
those skilled in the art or by any similar method yet to be
disclosed. One example is the determination of the IC.sub.50 (half
maximal inhibitory concentration) of the pharmaceutical composition
in vitro in cell lines or target molecules. Another example is the
determination of the LD.sub.50 (lethal dose causing death in 50% of
the tested animals) of the pharmaceutical composition in
experimental animals. The exact techniques used in determining an
effective amount will depend on factors such as the type and
physical/chemical properties of the pharmaceutical composition, the
property being tested, and whether the test is to be performed in
vitro or in vivo. The determination of an effective amount of a
pharmaceutical composition will be well known to one of skill in
the art who will use data obtained from any tests in making that
determination. Determination of an effective amount of disclosed
compound for addition to a cancer cell also includes the
determination of an effective therapeutic amount, including the
formulation of an effective dose range for use in vivo, including
in humans.
[0089] Treatment is contemplated in living entities including but
not limited to mammals (particularly humans) as well as other
mammals of economic or social importance, including those of an
endangered status. Further examples include livestock or other
animals generally bred for human consumption and domesticated
companion animals.
[0090] The toxicity and therapeutic efficacy of a pharmaceutical
composition may be determined by standard pharmaceutical procedures
in cell cultures or animals. Examples include the determination of
the IC.sub.50 (the half maximal inhibitory concentration) and the
LD.sub.50 (lethal dose causing death in 50% of the tested animals)
for a subject compound. The data obtained from these cell culture
assays and animal studies can be used in formulating a range of
dosage for use in human. The dosage may vary depending upon the
dosage form employed and the route of administration utilized.
[0091] The effective amount of the disclosed compound to results in
the slowing of expansion of the cancer cells would preferably
result in a concentration at or near the target tissue that is
effective in slowing cellular expansion in cancer cells, but have
minimal effects on non-cancer cells, including non-cancer cells
exposed to radiation or recognized chemotherapeutic chemical
agents. Concentrations that produce these effects can be determined
using, for example, apoptosis markers such as the apoptotic index
and/or caspase activities either in vitro or in vivo.
[0092] Treatment of a condition is the practice of any method,
process, or procedure with the intent of halting, inhibiting,
slowing or reversing the progression of a disease, disorder or
condition, substantially ameliorating clinical symptoms of a
disease disorder or condition, or substantially preventing the
appearance of clinical symptoms of a disease, disorder or
condition, up to and including returning the diseased entity to its
condition prior to the development of the disease.
[0093] The addition of a therapeutically effective amount of the
disclosed compound encompasses any method of dosing of a compound.
Dosing of the disclosed compound may include single or multiple
administrations of any of a number of pharmaceutical compositions
that include the disclosed compound as an active ingredient.
Examples include a single administration of a slow release
composition, a course of treatment involving several treatments on
a regular or irregular basis, multiple administrations for a period
of time until a diminution of the disease state is achieved,
preventative treatments applied prior to the instigation of
symptoms, or any other dosing regimen known in the art or yet to be
disclosed that one skilled in the art would recognize as a
potentially effective regimen. A final dosing regimen including the
regularity of and mode of administration will be dependent on any
of a number of factors including but not limited to the subject
being treated; the severity of the affliction; the manner of
administration, the stage of disease development, the presence of
one or more other conditions such as pregnancy, infancy, or the
presence of one or more additional diseases; or any other factor
now known or yet to be disclosed that affects the choice of the
mode of administration, the dose to be administered and the time
period over which the dose is administered.
[0094] Pharmaceutical compositions that include the disclosed
compound may be administered prior to, concurrently with, or after
administration of a second pharmaceutical composition that may or
may not include the compound. If the compositions are administered
concurrently, they are administered within one minute of each
other. If not administered concurrently, the second pharmaceutical
composition may be administered a period of one or more minutes,
hours, days, weeks, or months before or after the pharmaceutical
composition that includes the compound Alternatively, a combination
of pharmaceutical compositions may be cyclically administered.
Cycling therapy involves the administration of one or more
pharmaceutical compositions for a period of time, followed by the
administration of one or more different pharmaceutical compositions
for a period of time and repeating this sequential administration,
in order to reduce the development of resistance to one or more of
the compositions, to avoid or reduce the side effects of one or
more of the compositions, and/or to improve the efficacy of the
treatment.
[0095] The invention further encompasses kits that facilitate the
administration of the disclosed compound to a diseased entity. An
example of such a kit includes one or more unit dosages of the
compound. The unit dosage would be enclosed in a preferably sterile
container and would be comprised of the disclosed compound and a
pharmaceutically acceptable carrier. In another aspect, the unit
dosage would comprise one or more lyophilates of the compound. In
this aspect of the invention, the kit may include another
preferably sterile container enclosing a solution capable of
dissolving the lyophilate. However, such a solution need not be
included in the kit and may be obtained separately from the
lyophilate. In another aspect, the kit may include one or more
devices used in administrating the unit dosages or a pharmaceutical
composition to be used in combination with the compound. Examples
of such devices include, but are not limited to, a syringe, a drip
bag, a patch or an enema. In some aspects of the invention, the
device comprises the container that encloses the unit dosage.
[0096] Pharmaceutical compositions including the disclosed compound
may be used in methods of treating cancer. Such methods involve the
administration of a therapeutic amount of a pharmaceutical
composition that includes the disclosed compound and/or a
pharmaceutically acceptable salt thereof to a mammal, preferably a
mammal in which a cancer has been diagnosed.
[0097] A therapeutic amount further includes the prevention of
progression of the cancer to a neoplastic, malignant or metastatic
state. Such preventative use is indicated in conditions known or
suspected of preceding progression to cancer, in particular, where
non- or precancerous cell growth consisting of hyperplasia,
metaplasia, or most particularly, dysplasia has occurred (for
review of such abnormal growth conditions, see Robbins and Angell,
1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp.
68-90, incorporated by reference). Hyperplasia is a form of
controlled cell proliferation involving an increase in cell number
in a tissue or organ, without significant alteration in structure
or activity. For example, endometrial hyperplasia often precedes
endometrial cancer and precancerous colon polyps often transform
into cancerous lesions. Metaplasia is a form of controlled cell
growth in which one type of adult or fully differentiated cell
substitutes for another type of adult cell. Metaplasia can occur in
epithelial or connective tissue cells. A typical metaplasia
involves a somewhat disorderly metaplastic epithelium. Dysplasia is
frequently a forerunner of cancer, and is found mainly in the
epithelia; it is the most disorderly form of non-neoplastic cell
growth, involving a loss in individual cell uniformity and in the
architectural orientation of cells. Dysplastic cells often have
abnormally large, deeply stained nuclei, and exhibit pleomorphism.
Dysplasia characteristically occurs where there exists chronic
irritation or inflammation, and is often found in the cervix,
respiratory passages, oral cavity, and gall bladder.
[0098] Alternatively or in addition to the presence of abnormal
cell growth characterized as hyperplasia, metaplasia, or dysplasia,
the presence of one or more characteristics of a transformed
phenotype or of a malignant phenotype, displayed in vivo or
displayed in vitro by a cell sample derived from a patient can
indicate the desirability of prophylactic/therapeutic
administration of the pharmaceutical composition that includes the
compound. Such characteristics of a transformed phenotype include
morphology changes, looser substratum attachment, loss of contact
inhibition, loss of anchorage dependence, protease release,
increased sugar transport, decreased serum requirement, expression
of fetal antigens, disappearance of the 250,000 dalton cell surface
protein, etc. Further examples include leukoplakia, featuring a
benign-appearing hyperplastic or dysplastic lesion of the
epithelium, or Bowen's disease, a carcinoma in situ. Both of theses
are pre-cancerous lesions indicative of the desirability of
prophylactic intervention. In another example, fibrocystic disease
including cystic hyperplasia, mammary dysplasia, adenosis, or
benign epithelial hyperplasia is indicates desirability of
prophylactic intervention.
[0099] In some aspects of the invention, use of the disclosed
compound may be determined by one or more physical factors such as
tumor size and grade or one or more molecular markers and/or
expression signatures that indicate prognosis and the likely
response to treatment with the compound. For example, determination
of estrogen (ER) and progesterone (PR) steroid hormone receptor
status has become a routine procedure in assessment of breast
cancer patients. See, for example, Fitzgibbons et al, Arch. Pathol.
Lab. Med. 124:966-78, 2000, incorporated by reference. Tumors that
are hormone receptor positive are more likely to respond to hormone
therapy and also typically grow less aggressively, thereby
resulting in a better prognosis for patients with ER+/PR+ tumors.
In a further example, overexpression of human epidermal growth
factor receptor 2 (HER-2/neu), a transmembrane tyrosine kinase
receptor protein, has been correlated with poor breast cancer
prognosis (see, e.g., Ross et al, The Oncologist 8:307-25, 2003),
and Her-2 expression levels in breast tumors are used to predict
response to the anti-Her-2 monoclonal antibody therapeutic
trastuzumab (Herceptin.RTM., Genentech, South San Francisco,
Calif.).
[0100] In another aspect of the invention, the diseased entity
exhibits one or more predisposing factors for malignancy that may
be treated by administration of a pharmaceutical composition
including the compound. Such predisposing factors include but are
not limited to chromosomal translocations associated with a
malignancy such as the Philadelphia chromosome for chronic
myelogenous leukemia and t (14; 18) for follicular lymphoma; an
incidence of polyposis or Gardner's syndrome that are indicative of
colon cancer; benign monoclonal gammopathy which is indicative of
multiple myeloma, kinship with persons who have had or currently
have a cancer or precancerous disease, exposure to carcinogens, or
any other predisposing factor that indicates in increased incidence
of cancer now known or yet to be disclosed.
[0101] The invention further encompasses methods of treating cancer
that comprise combination therapies that comprise the
administration of a pharmaceutical composition including the
disclosed compound and another treatment modality. Such treatment
modalities include but are not limited to, radiotherapy,
chemotherapy, surgery, immunotherapy, cancer vaccines,
radioimmunotherapy, treatment with pharmaceutical compositions
other than those which include the disclosed compound, or any other
method that effectively treats cancer in combination with the
disclosed compound now known or yet to be disclosed. Combination
therapies may act synergistically. That is, the combination of the
two therapies is more effective than either therapy administered
alone. This results in a situation in which lower dosages of both
treatment modality may be used effectively. This in turn reduces
the toxicity and side effects, if any, associated with the
administration either modality without a reduction in efficacy.
[0102] In another aspect of the invention, the pharmaceutical
composition including the disclosed compound is administered in
combination with a therapeutically effective amount of
radiotherapy. The radiotherapy may be administered concurrently
with, prior to, or following the administration of the
pharmaceutical composition including the compound. The radiotherapy
may act additively or synergistically with the pharmaceutical
composition including the compound. This particular aspect of the
invention would be most effective in cancers known to be responsive
to radiotherapy. Cancers known to be responsive to radiotherapy
include, but are not limited to, Non-Hodgkin's lymphoma, Hodgkin's
disease, Ewing's sarcoma, testicular cancer, prostate cancer,
ovarian cancer, bladder cancer, larynx cancer, cervical cancer,
nasopharynx cancer, breast cancer, colon cancer, pancreatic cancer,
head and neck cancer, esophogeal cancer, rectal cancer, small-cell
lung cancer, non-small cell lung cancer, brain tumors, other CNS
neoplasms, or any other such tumor now known or yet to be
disclosed.
[0103] Examples of pharmaceutical compositions that may be used in
combination with the disclosed compound may include nucleic acid
binding compositions such as cis-diamminedichloro platinum (II)
(cisplatin), doxorubicin, 5-fluorouracil, taxol, and topoisomerase
inhibitors such as etoposide, teniposide, irinotecan and topotecan.
Still other pharmaceutical compositions include antiemetic
compositions such as metoclopromide, domperidone, prochlorperazine,
promethazine, chlorpromazine, trimethobenzamide, ondansetron,
granisetron, hydroxyzine, acethylleucine monoethanolamine,
alizapride, azasetron, benzquinamide, bietanautine, bromopride,
buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol,
dolasetron, meclizine, methallatal, metopimazine, nabilone,
oxyperndyl, pipamazine, scopolamine, sulpiride,
tetrahydrocannabinols, thiethylperazine, thioproperazine and
tropisetron.
[0104] Still other examples of pharmaceutical compositions that may
be used in combination with the pharmaceutical composition
including the disclosed compound are hematopoietic colony
stimulating factors. Examples of hematopoietic colony stimulating
factors include, but are not limited to, filgrastim, sargramostim,
molgramostim and epoietin alfa. Alternatively, the pharmaceutical
composition including the disclosed compound may be used in
combination with an anxiolytic agent. Examples of anxiolytic agents
include, but are not limited to, buspirone, and benzodiazepines
such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam,
chlordiazepoxide and alprazolam.
[0105] Pharmaceutical compositions that may be used in combination
with pharmaceutical compositions that include the disclosed
compound may include analgesic agents. Such agents may be opioid or
non-opioid analgesic. Non-limiting examples of opioid analgesics
include morphine, heroin, hydromorphone, hydrocodone, oxymorphone,
oxycodone, metopon, apomorphine, normorphine, etorphine,
buprenorphine, meperidine, lopermide, anileridine, ethoheptazine,
piminidine, betaprodine, diphenoxylate, fentanil, sufentanil,
alfentanil, remifentanil, levorphanol, dextromethorphan,
phenazocine, pentazocine, cyclazocine, methadone, isomethadone and
propoxyphene. Suitable non-opioid analgesic agents include, but are
not limited to, aspirin, celecoxib, rofecoxib, diclofinac,
diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen,
ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid,
nabumetone, naproxen, piroxicam, sulindac or any other analgesic
now known or yet to be disclosed.
[0106] In other aspects of the invention, pharmaceutical
compositions including the disclosed compound may be used in
combination with a method that involves treatment of cancer ex
vivo. One example of such a treatment is an autologous stem cell
transplant. In this method, a diseased entity's autologous
hematopoietic stem cells are harvested and purged of all cancer
cells. A therapeutic amount of a pharmaceutical composition
including the disclosed compound may then be administered to the
patient prior to restoring the entity's bone marrow by addition of
either the patient's own or donor stem cells.
[0107] Cancers that may be treated by pharmaceutical compositions
including the disclosed compound either alone or in combination
with another treatment modality include solid tumors such as
fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon cancer, colorectal cancer, kidney cancer, pancreatic cancer,
bone cancer, breast cancer, ovarian cancer, prostate cancer,
esophageal cancer, stomach cancer, oral cancer, nasal cancer,
throat cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms'tumor, cervical cancer, uterine cancer, testicular
cancer, small cell lung carcinoma, bladder carcinoma, lung cancer,
epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma,
medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,
skin cancer, melanoma, neuroblastoma, and retinoblastoma.
[0108] Additional cancers that may be treated by pharmaceutical
compositions including the disclosed compound include blood borne
cancers such as acute lymphoblastic leukemia ("ALL,"), acute
lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia,
acute myeloblastic leukemia ("AML"), acute promyelocytic leukemia
("APL"), acute monoblastic leukemia, acute erythroleukemic
leukemia, acute megakaryoblastic leukemia, acute myelomonocytic
leukemia, acute nonlymphocyctic leukemia, acute undifferentiated
leukemia, chronic myelocytic leukemia ("CML"), chronic lymphocytic
leukemia ("CLL"), hairy cell leukemia, multiple myeloma,
lymphoblastic leukemia, myelogenous leukemia, lymphocytic leukemia,
myelocytic leukemia, Hodgkin's disease, non-Hodgkin's Lymphoma,
Waldenstrom's macroglobulinemia, Heavy chain disease, and
Polycythemia vera.
[0109] Examples that represent different aspects of the invention
follow. Such examples should not be construed as limiting the scope
of the disclosure. Alternative mechanistic pathways and analogous
structures within the scope of the invention would be apparent to
those skilled in the art.
[0110] The invention encompasses inhibitors of cell migration
activity and inhibitors of effector recruitment activity.
Inhibition encompasses any action that hinders, from any detectable
level up to and including complete inactivation, the progression of
a biological process. Such biological processes include expression
of a gene or activities of a gene product, progression of a
disease, normal and abnormal metabolic activities, interactions
between entities within an organism, or interactions between one
organism and another. Further nonlimiting examples of biological
processes include development, death, maturation, infection, pain,
apoptosis, or homeostasis. Inhibition includes actions that silence
or repress the expression of a gene. Inhibition also includes
actions that hinder the activity of the RNA product, protein
product, or postranslationally modified protein product of a gene.
Inhibition may be effectuated through a single agent that
inactivates a single gene or gene product, by a single agent that
inactivates a combination of more than one gene or gene product, a
combination of agents that inactivates a single gene or gene
product or a combination of agents that inactivates a combination
of more than one gene or gene product.
[0111] Inhibition may be effectuated directly by an agent that
directly causes the inhibition of a biological process or by agents
that trigger one or more different biological processes to
effectuate the inhibition of the first biological process. Agents
that cause inhibition may also be called inhibitors. Examples of
inhibitors include compositions such as compounds that trigger RNAi
silencing such as microRNA or siRNA, small molecular compounds,
proteins such as soluble receptors or antibodies or any fragment
thereof, including an Fab, F(ab).sub.2, Fv, scFv, Fc, phage display
antibody, peptibody or any other composition of matter that may
inactivate or hinder a biological process. Further nonlimiting
examples of inhibitors include X-rays, UV rays, visible light
including laser light, and sound.
[0112] Cell migration activity includes any mode through which a
cell may move in two-dimensional or three-dimensional space. Such
migration includes movement through the use of pseudopodia
including the adhesion of pseudopodia to a surface, a flagellum, a
cilium, acts of amoeboid movement, extravasation, myosin-actin
interactions, microtubule extension, or any other process through
which a cell moves itself from one place to another or changes its
morphology. In one aspect of the invention, cell migration activity
is measured through cell adhesion. Using adhesion, cell migration
activity may be measured by cell-cell aggregation, monolayer radial
migration, including adhesion to a cell matrix comprising laminin,
BSA or any other cell matrix component, three dimensional spheroid
dispersion, or any other method that measures adhesion based
cellular migration in space. Migration activity may be measured by
any method that detects that a cell has moved from one place to
another or has changed its morphology. Such methods include flow
cytometry, capillary electrophoresis, visual examination by light,
fluorescence, or electron microscopy, or any such method known in
the art or yet to be developed. Inhibitors of cell migration
activity are agents that disrupt any molecular or cellular process
involved in cell migration activity.
[0113] Effector recruitment activity includes any activity of a
protein that contributes to the formation of a complex of two or
more molecules that serves to catalyze one or more chemical
reactions. Effectors include any protein, nucleic acid or other
molecule that may be included in a complex that performs one or
more biological activities. Recruitment activity encompasses any
protein-protein interaction including phosphorylation,
dephosphorylation and other enzymatic activities, adhesion,
signaling cascades, and cytokine/chemokine interactions, any
protein-nucleic acid interactions, such as any of those involved in
transcription, translation or DNA replication, or any other process
that includes a protein interacting with another molecule.
Inhibitors of effector recruitment activity may disrupt the
interaction of a molecule with any of the proteins listed above,
the interaction between any of those proteins with each other, and
further includes any members of a complex that might be later
identified.
[0114] In one aspect of the invention, inhibitors of effector
recruitment activity may be identified on the basis of their
ability to disrupt the binding of a molecule to one or more of its
effectors. This specific binding may be measured by any method that
allows the measurement of a protein-protein interaction known in
the art. Such method include the following examples, alone or in
combination as necessary: co-immunoprecipitation, biomolecular
fluorescence complementation, fluorescence resonance energy
transfer, label transfer, a yeast two-hybrid screen, in-vivo
crosslinking, tandem affinity purification, chemical crosslinking,
quantitative immunoprecipitation combined with knock-down (QUICK),
dual polarization interferometry, protein-protein docking, static
light scattering, immunoprecipitation plus mass-spectrometry,
Strep-protein interaction experiment (SPINE), surface plasmon
resonance, fluorescence correlation spectroscopy, or any other
method of measuring the specific interaction between one protein
and another now known in the art or yet to be disclosed.
[0115] In another aspect of the invention a glioblastoma patient is
treated by first assessing the expression of a target and then
treating with an effective dose of an inhibitor of that target,
potentially in combination with Temozolimide. The effective dose of
a compound is that amount effective to prevent occurrence of the
symptoms of a disorder or to treat some symptoms of the disorder
from which the patient suffers. Effective dose also includes an
effective amount, a therapeutic amount, or any amount sufficient to
elicit the desired pharmacological or therapeutic effects, thus
resulting in effective prevention or treatment of the disorder.
Thus, when treating a patient with glioblastoma, an effective
amount of compound is an amount sufficient to slow, or arrest the
progression, migration, metastasis, growth, or development of the
tumor with the result that life is extended. Prevention includes a
delay in onset of symptoms. Treatment includes a decrease in the
symptoms associated with the disorder or an amelioration of the
recurrence of the symptoms of the disorder. A pharmacologically
acceptable dose encompasses any dose that may be administered to a
patient that will not be lethal to the patient or cause effects
that threaten the health or the life of the patient.
[0116] Patients include any human being, nonhuman primate,
companion animal, or mammal suffering from a disease. In one aspect
of the invention, the patient has symptoms that signify the
presence of a tumor or other growth in the brain. Such symptoms
include headache, seizures, mental or personality changes, mass
effect, or one of a number of focal or localized systems including
ringing or buzzing sounds, hearing loss, loss of coordination,
reduced sensation, weakness or paralysis, difficulty with walking
or speech, difficulty keeping balance, decreased muscle control, or
double vision. Patients may display one or more different brain
tumor types including acoustic neurinoma, astrocytoma, ependyoma,
glioblastoma multiforme, meningioma, metastatic tumors originating
from another tumor type, mixed glioblastoma,
oligodendroglioblastoma, or pineal region tumor.
Example
[0117] Elements and acts in the example are intended to illustrate
the invention for the sake of simplicity and have not necessarily
been rendered according to any particular sequence or embodiment.
The example is also intended to establish possession of the
invention by the Inventors.
[0118] Members of the TNFR superfamily (TNFRSF), most notably
TNFR1, have been shown to play a role in inducing cell invasion and
migration in several cancer types. An expression microarray
database containing 195 clinically annotated brain tumor specimens
publicly available at NCBI's Gene Expression Omnibus as dataset
GSE4290 was analyzed. Snap-frozen specimens from epileptogenic foci
(NB, n=24), low-grade astrocytomas (LGA, n=29), and glioblastoma
multiforme (GBM, n=82) with clinical information were collected at
the Hermelin Brain Tumor Center, Henry Ford Hospital (Detroit,
Mich.) as previously described (See Reference 24). Gene expression
profiling was conducted on all samples using Affymetrix U133 Plus 2
GeneChips according to the manufacturer's protocol at the
Neuro-Oncology Branch at the National Cancer Institute (Bethesda,
Md.). For the analysis, gene expression data were normalized in two
ways: per chip normalization and per gene normalization across all
samples in the collection. For per chip normalization, all
expression data on a chip were normalized to the 50th percentile of
all values on that chip. For per gene normalization, the data for a
given gene were normalized to the median expression level of that
gene across all samples. Gene expression differences were deemed
statistically significant using parametric tests where variances
were not assumed equal (Welch analysis of variance). Expression
values were then filtered for highly variable (differentially
expressed) genes (coefficient of variation >30%) across samples
producing a list of 7322 genes. TROY/TNFRSF19 expression is
significantly differentially expressed among brain specimens. In
normal brain specimens, TROY expression is relatively low, but is
increased with increasing tumor grade and is significantly higher
in GBM samples (n=82) (See FIG. 1). Quantitative RT-PCR was
performed on independent non-neoplastic (n=10), LGA (n=6),
anaplastic astrocytoma (n=4), and GBM (n=22) specimens. Normal
brain specimens show relatively low mRNA levels for TROY as
compared to the brain tumor samples (p<0.01). In GBM specimens,
the mRNA level of TROY is significantly higher than in normal brain
(p<0.01) (See FIG. 2). Next, principal component analysis was
done to discern possible relationships between subgroups of samples
as in, for example, Reference 34. Kaplan-Meier survival curves were
developed for each principal component cluster. One cluster had a
median survival time of 401 days (short-term survival, ST) and the
other cluster had a median survival time of 952 days (long-term
survival, LT). Box-and-whisker plots for TROY expression level in
each cluster derived from PC analysis were graphed. Significance
between the two populations was tested with a two-sample t-test
assuming unequal variances. Analysis of the Affymetrix expression
values for TROY in the GBM specimens for each cluster showed that
patients with GBM in the short-term survival cluster had higher
expression of TROY (10.5) than GBM patients in the long term
survival cluster (2.9; p<0.01) (See FIG. 3) This demonstrates
that high TROY expression levels correlates with poor patient
outcome while low TROY expression level corresponds with good
patient outcome.
[0119] TROY is expressed in glioblastoma cell lines and
siRNA-mediated depletion of TROY suppresses glioblastoma cell
migration. The expression of TROY protein was assessed in four
different cultured glioblastoma cell lines. The highest level of
expression of TROY was seen in U118 cells, the next highest level
of expression was seen in U87 cells, and the lowest level of
expression was seen in T98G and SNB19 cells (See FIG. 4).
[0120] RNAi was used to suppress the expression of TROY in each of
the four listed glioma cell lines and the migratory behavior of the
cells on glioma-derived ECM was examined using a two-dimensional
radial cell migration assay (See References 26 and 27). Suppression
of TROY protein expression in all glioma cell lines was
.about.80-90% effective with each of two independent siRNA
oligonucleotides. Representative results are shown for U118 cells
(See FIG. 5). Further, suppression of TROY expression by siRNA
resulted in a significant (p<0.05) inhibition of cell migration
in all four cell lines (See FIG. 18).
[0121] T98G and SNB19 glioma cell lines that stably express of
HA-epitope tagged TROY were produced through lentiviral
transduction. These were used to further examine the role TROY
signaling in glioma cell migration (See FIG. 7). Both the T98G and
SNB19 lines normally express low levels of endogenous TROY. The
cell lines with HA tagged-TROY showed a .about.1.8-2.3-fold
increase in cell migration rate (See FIG. 8). Migration of the
HA-tagged TROY expressing cells was further tested in the context
of an authentic brain microenvironment using an ex vivo organotypic
rat brain slice model. T98G glioma cells that overexpressed TROY
displayed a two-fold increase in the depth of cell invasion after
48 hours relative to controls (See FIG. 9). Immunolocalization of
TROY using an anti-HA antibody revealed that TROY was localized
near the cell perimeter and was enriched in lamellipodia (See FIG.
10 panel b).
[0122] Potential effector molecules of TROY were found in
immunoprecipitation experiments coupled with MALDI-TOF MS analysis.
In one experiment, T98G cells expressing HA-tagged TROY and control
T98G cells transfected with GFP were lysed, immunoprecipitated with
anti-HA antibodies, and the immunoprecipitates resolved by
SDS-PAGE. Prominent protein bands present in the immunoprecipitates
of TROY expressing cells but absent in the immunoprecipitates of
control cells of interest were recovered from the gel. Proteins
were eluted, and trypsin-digested. MALDI-TOF and MS-MS analysis of
the trypsin digests were performed on a Voyager reflector
instrument (Applied Biosystems) and a Q-STAR mass spectrometer
(Perceptive Biosystems) in positive ion mode.
[0123] The non-receptor protein tyrosine kinase Pyk2 was a
candidate sequence identified by mass spectrometry in the TROY
immunoprecipitate. Association of TROY with Pyk2 was verified by
co-immunoprecipitation. T98G cells transfected with HA-tagged TROY
or co-transfected with HA-tagged TROY and Pyk2 were
immunoprecipitated with anti-HA antibodies and the precipitates
immunoblotted with anti-Pyk2 antibodies (See FIG. 11). Both
endogenous Pyk2 and transfected Pyk2 co-immunoprecipitated with
TROY substantiating the intracellular interaction between TROY and
Pyk2.
[0124] Depletion of Pyk2 expression by shRNA in TROY overexpressing
T98G cells was performed to determine whether the association with
Pyk2 was required for TROY-induced stimulation of glioma migration.
Suppression of Pyk2 expression by shRNA significantly inhibited
TROY stimulated glioma cell migration (See FIG. 12). Further,
coexpression of a kinase inactive variant of Pyk2 (Pyk2KD) with
TROY HA significantly inhibited the migration of the control T98G
cells indicating that Pyk2 activity is required for TROY stimulated
migration of glioma cells. (See FIG. 13). Finally, silencing of
Pyk2 expression also inhibited TROY mediated Rac1 activation (See
FIG. 15). Together, these results indicate that TROY-mediated
glioma cell migration is dependent upon Pyk2 activity.
[0125] Rho GTPase family members, particularly Rac1 (See References
22-24, 28) effect the invasive behavior of glioblastoma cells. As a
result, if TROY signaling influences Rac1 activity, then TROY is a
marker of invasive glioblastoma and Rac1 is an effector molecule of
TROY. U118 cells express a high endogenous level of TROY protein
expression (See FIG. 4) and display high Rac1 activity (See FIG.
14). Reduction of TROY expression in U118 cells by siRNA resulted
in decreased activity of Rac1 (See FIG. 14). Further, siRNA
mediated reduction of TROY expression induced RhoA activation,
showing that TROY signaling modulates Rac1 and RhoA GTPases
activity in opposite directions. Indeed, it has been previously
noted that in certain cell types, overexpression of TROY increased
RhoA activation (See References 6 and 9) suggesting that TROY
signaling may be modulated by cell type specific elements. To
validate the effect of TROY on Rac1 activity, the activation of
Rac1 in glioma cells overexpressing TROY was compared to the
activation of Rac1 in untransfected cells. Overexpression of TROY
resulted in a .about.2-fold induction of Rac1 activation relative
to untransfected cells (See FIG. 15).
[0126] Since Pyk2 interacts with TROY and mediates TROY-induced
migration, the effect of Rac1 activation induced by TROY expression
is dependent upon Pyk2 activity was determined. shRNA-mediated
depletion of Pyk2 in TROY overexpressing glioma cells suppressed
TROY induced Rac1 activity to the level of that in control cells.
(See FIG. 15). This indicates that the TROY-mediated regulation of
Rac1 activation is dependent upon Pyk2. Further, Rac1 expression in
T98G cells overexpressing the TROY receptor was reduced by Rac1
siRNA. That reduction in Rac1 expression in was .about.90%
effective in T98G cells and caused a significant inhibition of
TROY-mediated cell migration. (See FIG. 16).
[0127] A recent study suggest that TROY is activated by the TNF
family ligand lymphotoxin-.alpha. to induce NF.kappa.B activation,
whereas previous studies have not revealed specific interactions
between TROY and any of the TNF family members (See Reference 29).
Since Rac1 can influence multiple downstream signaling pathways,
immunoblot analysis of lysates from TROY overexpressing cells were
analyzed for detection of various signaling pathways and compared
to lysates from untransfected cells. Increased phosphorylation of
Akt, I.kappa.B.alpha., and ERK1/2 in TROY overexpressing cells was
observed relative to untransfected cells (See FIG. 18).
[0128] Activation of Akt and NF.kappa.B signaling pathways plays a
critical role in cell survival. The effect of TROY expression on
chemotherapy-induced apoptosis in glioma cells was then determined
by comparing. the sensitivity of control U118 glioma cells and U118
glioma cells reduced TROY expression by transfection of TROY
specific RNAi to temozolomide treatment. U118 cells with reduced
expression of TROY were significantly more sensitive to cell death
following temozolomide treatment relative to U118 cells transfected
with a negative RNAi control (See FIG. 19). Conversely, T98G glioma
cells overexpressing TROY were significantly more resistant to
temozolomide induced apoptosis relative to control transfected T98G
glioma cells (See FIG. 20). Together, these data indicate that TROY
stimulated glioma cell migration/invasion increases resistance to
chemotherapy-induced cell death in glioma.
[0129] A number of RhoGTPases, including Rac1 Rac3 and Cdc42 (See
References 22-25), contribute to glioblastoma cell invasion in
vitro. The Rho GTPases are activated by GEFs. There are currently
80 RhoGEFs in the human genome. Of these GEFs, 26 are known Rac1
activators, and currently, it is not known which Rac GEFs
contribute to Rac1 activity in glial tumors. Rac GEFs that mediate
glioma invasion were identified by first mining the NCBI expression
microarray database of human brain tumor specimens for the 26 GEFs
known to have Rac exchange factor activity. Of the 26 GEFs, Ect2,
Trio and Vav3 exhibited increased expression in glioblastomas
(GBMs) versus normal brain. Depletion of Ect2, Trio, and Vav3
expression reduced Rac1 activity in glioblastoma cells which in
turn led to a subsequent inhibition of glioblastoma cell migration
and invasion. A library of small interfering RNAs (siRNAs) directed
against all 26 Rac GEFs in the human genome was used to evaluate
the role of RacGEF's in inhibiting glioma invasion in a 96-well
format invasion assay. Two additional Rac GEFs--Dock180 and
Dock7--were found to contribute to glioma invasion. Knockdown of
Dock180 or Dock7 expression by RNAi significantly reduced glioma
invasion in vitro (See FIG. 21). Dock180 has recently been reported
(See Reference 30) to be overexpressed in invasive glioma cells
where it regulates Rac1 activity and glioma cell invasion. To
further examine the role of Dock7 in glioblastoma cell invasion
using RNAi sequences that inhibit Dock 7 expression to inhibit the
migration of SNB19 cells into rat brain slices, a well-established
ex vivo organotypic model for glioma invasion. Knockdown of Dock7
expression significantly inhibited invasion relative to control
cells (See FIG. 22).
[0130] A significant limitation of the use of long-term established
human glioma cell lines for orthotopic xenografts is their
propensity to form discrete, non-invasive tumors with well
circumscribed borders that push into the adjacent normal brain
tissue (See References 31-33). This is in contrast to the diffuse
highly infiltrative growth that defines primary GBM in patients.
More important is the loss of genetic features and signatures in
long-term established cell lines which are common to primary GBM. A
model based on utilizing primary glioma xenografts established and
maintained by direct heterotypic transplantation, propagation, and
passaging of patient tumor surgical samples in immune deficient
mice has been established. Intracranial tumors established with
these GBM xenografts retain key histopathological characteristics
of the aggressive behavior of the patients' tumors including local
invasion at the tumor periphery and invasion along white matter
tracks, as well as manifesting key genetic features such as
preservation of EGFR amplification status. Therefore, tumors that
arise from these xenograft lines adequately model primary GBM in
patients (See References 34-36). TROY protein expression was
examined in lysates obtained from 19 xenografts grown
orthotopically in murine brain. Examination of TROY expression on
the xenograft lysates showed a range of TROY expression.
Representative immunoblots showing GBM xenografts with high TROY
expression (GBM10), intermediate levels of TROY expression (GBM6,
GBM8), or low levels of TROY expression (GBM44, GBM46) are shown in
FIG. 23.
[0131] Referring now to FIG. 1: TROY mRNA expression levels derived
from the NCBI Gene Expression Omnibus GDS1962 dataset are presented
as box-and-whisker plots. The box for each gene indicates the
interquartile range (25-75th percentile) and the line within this
box indicates the median value. Bottom and top bars of the whisker
indicate the 10th and 90th percentiles, respectively. Outliers are
represented by closed circles. Significance between the indicated
classes of brain specimens was tested using a two-sample t test
assuming unequal variances. (NB=non-neoplastic brain; OL,
Oligodendrogliomas; Astro=low grade astrocytomas; GBM=glioblastoma
multiforme). Referring now to FIG. 2 Quantitative real-time PCR
analysis of TROY expression in non-neoplastic brain (NB), grade 1
low grade astrocytoma (LGA), grade 2-3 Astrocytomas (Astro) and
glioblastoma multiforme (GBM) indicates that a higher level of TROY
expression signifies increased tumor grade. Values were normalized
to histone H3.3 and HPRT1 reference genes. Data are presented as
box-and-whisker plots. Referring now to FIG. 3: principal component
analysis of brain tumors from NCBI Gene Expression Omnibus GDS1962
dataset revealed two groups differing by their survival and were
denoted as long term (LT) survival and short-term (ST) survival.
These indicate that a higher level of TROY expression signifies an
association with short-term survival. Box-and-whisker plots for
TROY expression in GBM specimens for each cluster are shown.
Significance between the two populations was tested with a
two-sample t test assuming unequal variances.
[0132] Referring now to FIG. 4: T98G, SNB19, U87, and U118 cell
lysates were analyzed for endogenous level of TROY expression by
immunoblotting. All express TROY with U87 and U118 cells having the
highest expression level. The levels of .alpha.-tubulin protein
were also immunoblotted to ensure equal sample loading. Referring
now to FIG. 5: knockdown of TROY expression in U118 cells by two
independent siRNA oligonucleotides. Note reduced expression of TROY
protein in TROY-1 and TROY-2 transfected U118 cells. Referring now
to FIG. 18: the migration rate of each of the four cell lines was
slowed when the cell lines were transfected with siRNA
oligonucleotides targeting TROY. siRNA targeting luciferase was
used as a negative control. Migration rate was determined after 24
h migration on glioma derived ECM (*-p<0.05).
[0133] Referring now to FIG. 7: Lysates of T98G or SNB19 cells
transduced with empty lentiviral vector (v) or lentiviral vector
encoding HA-epitope tagged TROY immunoblotted with anti-HA antibody
shows that TROY is overexpressed in cell lines transfected with the
TROY construct. Referring now to FIG. 8: the migration rate of
HA-TROY expressing glioma cells is faster than that of cells
transduced with a negative control construct. Cell migration was
assessed over 48 h. Data represents the average of three
independent experiments (*, p<0.01; **, p<0.05). Referring
now to FIG. 9: T98G cells stably expressing green fluorescent
protein were transduced with lentiviruses expressing HA-tagged
TROY. Cells were implanted into the bilateral putamen on rat
organotypic brain slices and observed at 48 h. Depth of invasion
was calculated from Z-axis images collected by confocal laser
scanning microscopy. The mean value of the depth of invasion was
obtained from six independent experiments (*, p<0.01). Cell
lines transduced with a construct containing TROY displayed
significantly greater depth of invasion. Referring now to FIG. 10:
immunofluorescent staining for Troy in T98G-Troy-HA cells using an
anti-HA antibody shows that TROY localizes at the membrane
periphery and within cellular extensions.
[0134] Referring now to FIG. 11: The top panel indicates that
lysates of negative control transfected T98G cells, T98G cells
transfected with HA-TROY, T98G cells transfected with Pyk2, or T98G
cells cotransfected with HA-TROY and Pyk2 show expression of the
transfected constructs when immunoblotted with anti-Pyk2 or anti-HA
antibodies. In the bottom panel, the same cell lines were
immunoprecipitated with anti-HA antibody and the precipitates
immunoblotted with anti-HA or anti-Pyk2 indicating that Pyk2
associates with TROY. Referring now to FIG. 12: The inhibition of
Pyk2 expression by RNAi targeting Pyk2 suppresses Troy-induced
glioma migration. Migration rate of T98G, T98G-Troy-HA, and
T98G-Troy-HA cells transfected with a shRNA targeting Pyk2 was
assessed over 24 h using a radial migration assay on 10 .mu.g/ml
laminin substrate (*, p<0.01). T98G cells overexpressing TROY
migrate at a faster rate than T98G cells that lack the TROY
expressing construct. This effect is negatived by transfection with
Pyk2-specific shRNA. Referring now to FIG. 13: inhibition of Pyk2
activity inhibits Troy-induced glioma migration. T98G or
T98G-Troy-HA expressing cells were infected with recombinant
adenoviruses expressing a Pyk2 variant lacking the Pyk2 kinase
domain. (Pyk2KD). Cell migration was assessed over 24 h using a
radial migration assay on 10 .mu.g/ml laminin substrate (*,
p<0.01). Transfection of the Pyk2KD construct into T98G cells
that do not overexpress TROY slowed the migration rate of those
cells. T98G cells overexpressing TROY migrate at an even faster
rate, but this effect is mitigated by transfection with Pyk2KD.
[0135] Referring now to FIG. 14: U118 cells were left untransfected
(NT), transfected with an siRNA targeting nonmammalian luciferase
(ctrl), or an siRNA targeting TROY (Troy-1). Cells were cultured
under serum-free medium for an additional 16 hr prior to RhoA and
Rac1 activation assays. Immunoblots show that RhoA is more likely
to be phosphorylated and Rac-1 is dephosphorylated upon suppression
of TROY1 expression. Referring now to FIG. 15: T98G and
T98G-Troy-HA cells were transfected with a shRNA targeting Pyk2 and
cultured under serum-free medium for 16 h. Lysates were then
analyzed for activation of Rac1. Overexpression of TROY leads to
more Rac1 phosphorylation. This effect is diminished when Pyk2
expression is suppressed. Referring now to FIG. 16: suppression of
Rac1 expression by siRNA suppresses Troy-induced glioma cell
migration. T98G and T98G-TROY-HA cells were transfected with an
siRNA oligonucleotide targeting Rac1. Cell migration was assessed
over 24 h using a radial migration assay on 10 mg/ml laminin
substrate (*, p<0.01). Suppression of Rac1 expression reduced
the migration rate of the cells indicating that TROY-1 mediated
migration works through Rac1. Referring now to FIG. 17: a Western
blot validating suppression of Rac1 and HA-TROY expression was
performed.
[0136] Referring now to FIG. 18: TROY overexpression induces
activation of Akt, NFkB and Erk1/2 signaling pathways. Cellular
lysates of T98G glioma cells or T98G cells overexpressing TROY
(left panel) and SNB19 glioma cells or SNB19 cells overexpressing
TROY (right panel) were immunoblotted with the indicated
antibodies. Equal sample loading was verified by immunoblotting
lysates with an anti-.alpha.-tubulin antibody. Western blots
indicate phosphorylation of Akt, IkB.alpha., and Erk1/2.
[0137] Referring now to FIG. 19: U118 cells were transfected with a
siRNA targeting TROY (Troy-1) or a siRNA targeting a nonmammalian
gene. Cells were then treated with 250 .mu.M of temozolomide (TMZ)
or vehicle (DMSO) for 48 h. The percentage of cell viability was
measured by Alamar Blue assay and normalized to the control siRNA
untreated with TMZ (*, p<0.01; **, p<0.001). The results
indicate that when TROY expression is suppressed, the cells are
rendered sensitive to temozolimide. Referring now to FIG. 20: T98G
and T98G expressing TROY-HA were treated with 250 .mu.M of TMZ or
vehicle (DMSO) for 48 h. The percentage of cellular apoptosis was
measured by annexin V staining followed by flow cytometry. Data
represents the mean and S.D. from three independent experiments
with each experiment conducted in triplicate (**, p<0.001). The
results show that overexpression of TROY decreases the number of
cells that apoptose upon treatment with temozolimide and that
overexpression of TROY increases resistance to temozolimide.
[0138] Five known Rac1 activators were assessed for their ability
to contribute to glioma invasion identified in a focused RhoGEF
genome-wide siRNA screening approach. Referring now to FIG. 21:
SNB19 cells transfected with siRNAs targeting luciferase (ctrl),
Dock180, or Dock7 were plated in transwell invasion chambers coated
with Matrigel, and 24 h later, cells that had migrated through the
filter were stained and counted. Shown are the mean of at least two
independent experiments; bars, .+-.SE (*, p<0.001). Results
indicate that Dock180 and Dock7 are implicated in the invasive
phenotype of glioma cells. Referring now to FIG. 22: SNB19-GFP
cells were transfected with a siRNA targeting luciferase (ctrl) or
two independent siRNAs targeting Dock7. After 24 h, cells were
implanted into the bilateral putamen on rat organotypic brain
slices and cultured for 48 h. Depth of invasion was calculated from
Z-axis images collected by confocal laser scanning microscopy. The
mean value of the depth of invasion (+/-SEM) was obtained from
three independent experiments, performed in triplicates (*,
p<0.01). The results further indicate a role of Dock7 in the
development of an invasive phenotype.
[0139] Referring now to FIG. 23: Total cellular lystates from
glioblastoma multiforme xenografts grown in murine brain
orthopically were collected and immunoblotted for human TROY.
Ponceau staining was used as a loading control.
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Sequence CWU 1
1
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tgtcaaccta gtgcctagtc ataattaatt gacttttcct 3240gtattacttc
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3300acattgcctt ttggacatgt aaaatattta ggatttgacc acacaatggc
tatgaaaatg 3360caagtagttt cctcgcgtga cctcaccatg attcacatac
gtgccactgt ttgaaatctg 3420gtctgtttgc atttctgtta tgacagagag
atgatgtttg catttctgtt atgacagaga 3480gatgatgaaa gtaggcaggg
ctgtgttcct ttgtgtagcc tgtatatatt ttccatatgt 3540agagccctga
ttaacttcaa ggacaaacac tggctggaga aagccagact gatgggaatg
3600agactttggc caaaaatccc aaaacatcat tttcaatcag tagagaagtg
cttagggttg 3660aaaattgatt tcatttgcta ctgaatttgg taaatcctgg
gtaactttta tcaagatgaa 3720gacattttac cctacctact ctagaaatat
acaacaatgt tatattttac actccttgga 3780aacatttgag gaaaaaaatg
caatttgcac ttcactttgt tggaatatcc catagcactc 3840aataaactca
gctgctagag tgccgatgtc aggagggctg tgtcgggtaa tgcgtgtggc
3900tgaatgggca taaccactgt ggcttcttgt gctgcagaag ctcgttgaca
agactgagga 3960ggatttcaag agcaaccaag taaagtcatg aattttctaa
ttttgtgtat atggaatata 4020tttttaaata gagatttttc tactttagat
aatgtgttaa tattgctatt acctaggtta 4080agcactattg tctgtgctag
taagaaaaag aaaggaaaac catcattgct ttatagtagc 4140ttatatcaat
ttagatttca tcattactat tttgcatact ggaatttata aatgtgtaaa
4200ttatcatttt cttagttttg taataccttt tttatttgtg aataaaatta
tcacctggta 4260ttcttaaaaa aaaaaaaaaa aaa 42832417PRTHomo Sapiens
2Met Ala Leu Lys Val Leu Leu Glu Gln Glu Lys Thr Phe Phe Thr Leu1 5
10 15Leu Val Leu Leu Gly Tyr Leu Ser Cys Lys Val Thr Cys Glu Ser
Gly 20 25 30Asp Cys Arg Gln Gln Glu Phe Arg Asp Arg Ser Gly Asn Cys
Val Pro 35 40 45Cys Asn Gln Cys Gly Pro Gly Met Glu Leu Ser Lys Glu
Cys Gly Phe 50 55 60Gly Tyr Gly Glu Asp Ala Gln Cys Val Thr Cys Arg
Leu His Arg Phe65 70 75 80Lys Glu Asp Trp Gly Phe Gln Lys Cys Lys
Pro Cys Leu Asp Cys Ala 85 90 95Val Val Asn Arg Phe Gln Lys Ala Asn
Cys Ser Ala Thr Ser Asp Ala 100 105 110Ile Cys Gly Asp Cys Leu Pro
Gly Phe Tyr Arg Lys Thr Lys Leu Val 115 120 125Gly Phe Gln Asp Met
Glu Cys Val Pro Cys Gly Asp Pro Pro Pro Pro 130 135 140Tyr Glu Pro
His Cys Ala Ser Lys Val Asn Leu Val Lys Ile Ala Ser145 150 155
160Thr Ala Ser Ser Pro Arg Asp Thr Ala Leu Ala Ala Val Ile Cys Ser
165 170 175Ala Leu Ala Thr Val Leu Leu Ala Leu Leu Ile Leu Cys Val
Ile Tyr 180 185 190Cys Lys Arg Gln Phe Met Glu Lys Lys Pro Ser Trp
Ser Leu Arg Ser 195 200 205Gln Asp Ile Gln Tyr Asn Gly Ser Glu Leu
Ser Cys Phe Asp Arg Pro 210 215 220Gln Leu His Glu Tyr Ala His Arg
Ala Cys Cys Gln Cys Arg Arg Asp225 230 235 240Ser Val Gln Thr Cys
Gly Pro Val Arg Leu Leu Pro Ser Met Cys Cys 245 250 255Glu Glu Ala
Cys Ser Pro Asn Pro Ala Thr Leu Gly Cys Gly Val His 260 265 270Ser
Ala Ala Ser Leu Gln Ala Arg Asn Ala Gly Pro Ala Gly Glu Met 275 280
285Val Pro Thr Phe Phe Gly Ser Leu Thr Gln Ser Ile Cys Gly Glu Phe
290 295 300Ser Asp Ala Trp Pro Leu Met Gln Asn Pro Met Gly Gly Asp
Asn Ile305 310 315 320Ser Phe Cys Asp Ser Tyr Pro Glu Leu Thr Gly
Glu Asp Ile His Ser 325 330 335Leu Asn Pro Glu Leu Glu Ser Ser Thr
Ser Leu Asp Ser Asn Ser Ser 340 345 350Gln Asp Leu Val Gly Gly Ala
Val Pro Val Gln Ser His Ser Glu Asn 355 360 365Phe Thr Ala Ala Thr
Asp Leu Ser Arg Tyr Asn Asn Thr Leu Val Glu 370 375 380Ser Ala Ser
Thr Gln Asp Ala Leu Thr Met Arg Ser Gln Leu Asp Gln385 390 395
400Glu Ser Gly Ala Val Ile His Pro Ala Thr Gln Thr Ser Leu Gln Glu
405 410 415Ala322DNAHomo Sapiens 3ggccaaaaat cccaaaacat ca
22422DNAHomo Sapiens 4ccacagtggt tatgcccatt ca 22
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